Functional analysis, overexpression, and kinetic characterization of pyruvate kinase from methicillin-resistant staphylococcus aureus

Article abstract of DOI:10.1021/bi100780t

Novel antimicrobial targets are urgently needed to overcome rising antibiotic resistance of important human pathogens including methicillin-resistant Staphylococcus aureus (MRSA). Here we report the essentiality and kinetic properties of MRSA pyruvate kinase (PK). Targetron-mediated gene disruption demonstrated PK is essential for S. aureus growth and survival, suggesting that this protein may be a potential drug target. The presence of the pfk (6-phosphofructokinase)-pyk operon in MRSA252, and the nonessential nature of PFK shown by targetron, further emphasized the essential role of PK in cell viability. The importance of PK in bacterial growth was confirmed by showing that its enzymatic activity peaked during the logarithmic phase of S. aureus growth. PK from Staphylococcus and several other species of bacteria have an extra C-terminal domain (CT) containing a phosphoenolpyruvate (PEP) binding motif. To elucidate the possible structure and function of this sequence, the quaternary structures and kinetic properties of the full-length MRSA PK and truncated MRSA PK lacking the CT domain were characterized. Our results showed that (1) MRSA PK is an allosteric enzyme with homotetramer architecture activated by AMP or ribose 5-phosphate (R5P), but not by fructose 1,6-bisphosphate (FBP), which suggests a different mode of allosteric regulation when compared with human isozymes, (2) the CT domain is not required for the tetramerization of the enzyme; homotetramerization occurred in a truncated PK lacking the domain, (3) truncated enzyme exhibited high affinity toward both PEP and ADP and exhibited hyperbolic kinetics toward PEP in the presence of activators (AMP and R5P) consistent with kinetic properties of full-length enzyme, indicating that the CT domain is not required for substrate binding or allosteric regulation observed in the holoenzyme, (4) the kinetic efficiency (k_cat/S_0.5) of truncated enzyme was decreased by 24- and 16-fold, in ligand-free state, toward PEP and ADP, respectively, but was restored by 3-fold in AMP-bound state, suggesting that the sequence containing the CT domain (Gly⁴⁷³-Leu⁵⁸⁵) plays a substantial role in enzyme activity and comformational stability, and (5) full-length MRSA PK activity was stimulated at low concentrations of ATP (e.g., 1 mM) and inhibited by inorganic phosphate and high concentrations of FBP (10 mM) and ATP (e.g., >2.5 mM), whereas for truncated enzyme, stimulation at low concentrations of ATP was lost. These findings suggest that the CT domain is involved in maintaining the specificity of allosteric regulation of MRSA PK by AMP, R5P, and ATP. The CT extension also encodes a protein domain with homology to enzyme I of the Escherichia coli sugar-PTS system, suggesting that MRSA PK may also exert an important regulatory role in sugar transport metabolism. These findings yield new insights into MRSA PK function and mode of allosteric regulation which may aid in the development of clinically important drugs targeting this enzyme and further define the role of the extra C-terminal domain in modulating the enzyme's activity.

Full text of DOI:10.1021/bi100780t

Biochemistry 2010, 49, 7733–7747 7733
DOI: 10.1021/bi100780t
Functional Analysis, Overexpression, and Kinetic Characterization of Pyruvate Kinase
from Methicillin-Resistant Staphylococcus aureus†
Roya Zoraghi,‡ Raymond H. See,‡,^ Huansheng Gong,‡ Tian Lian,‡ Rick Swayze,‡ B. Brett Finlay,§,
Robert C. Brunham,‡,^ William R. McMaster,‡, and Neil E. Reiner*,‡,
§
‡Division of Infectious Diseases, Department of Medicine, Department of Biochemistry and Molecular Biology, Department of
Microbiology and Immunology, and ^Centre for Disease Control, University of British Columbia, Vancouver, Britsih Columbia, Canada V5Z 3J5
Received May 18, 2010; Revised Manuscript Received July 29, 2010
ABSTRACT: Novel antimicrobial targets are urgently needed to overcome rising antibiotic resistance of
important human pathogens including methicillin-resistant Staphylococcus aureus (MRSA). Here we report
the essentiality and kinetic properties of MRSA pyruvate kinase (PK). Targetron-mediated gene disruption
demonstrated PK is essential for S. aureus growth and survival, suggesting that this protein may be a potential
drug target. The presence of the pfk (6-phosphofructokinase)-pyk operon in MRSA252, and the nonessential
nature of PFK shown by targetron, further emphasized the essential role of PK in cell viability. The
importance of PK in bacterial growth was confirmed by showing that its enzymatic activity peaked during the
logarithmic phase of S. aureus growth. PK from Staphylococcus and several other species of bacteria have an
extra C-terminal domain (CT) containing a phosphoenolpyruvate (PEP) binding motif. To elucidate the
possible structure and function of this sequence, the quaternary structures and kinetic properties of the full-
length MRSA PK and truncated MRSA PK lacking the CT domain were characterized. Our results showed
that (1) MRSA PK is an allosteric enzyme with homotetramer architecture activated by AMP or ribose
5-phosphate (R5P), but not by fructose 1,6-bisphosphate (FBP), which suggests a different mode of allosteric
regulation when compared with human isozymes, (2) the CT domain is not required for the tetramerization of
the enzyme; homotetramerization occurred in a truncated PK lacking the domain, (3) truncated enzyme
exhibited high affinity toward both PEP and ADP and exhibited hyperbolic kinetics toward PEP in the
presence of activators (AMP and R5P) consistent with kinetic properties of full-length enzyme, indicating that
the CT domain is not required for substrate binding or allosteric regulation observed in the holoenzyme, (4)
the kinetic efficiency (kcat/S0.5) of truncated enzyme was decreased by 24- and 16-fold, in ligand-free state,
toward PEP and ADP, respectively, but was restored by 3-fold in AMP-bound state, suggesting that the
sequence containing the CT domain (Gly473-Leu585) plays a substantial role in enzyme activity and
comformational stability, and (5) full-length MRSA PK activity was stimulated at low concentrations of
ATP (e.g., 1 mM) and inhibited by inorganic phosphate and high concentrations of FBP (10 mM) and ATP
(e.g., >2.5 mM), whereas for truncated enzyme, stimulation at low concentrations of ATP was lost. These
findings suggest that the CT domain is involved in maintaining the specificity of allosteric regulation of
MRSA PK by AMP, R5P, and ATP. The CT extension also encodes a protein domain with homology to
enzyme I of the Escherichia coli sugar-PTS system, suggesting that MRSA PK may also exert an important
regulatory role in sugar transport metabolism. These findings yield new insights into MRSA PK function and
mode of allosteric regulation which may aid in the development of clinically important drugs targeting this
enzyme and further define the role of the extra C-terminal domain in modulating the enzyme’s activity.
Pyruvate kinase (PK)1 (EC 2.7.1.40), a final-stage enzyme in
glycolysis, catalyzes the transfer of a phosphoryl group from
phosphoenolpyruvate (PEP) to adenosine diphosphate (ADP),
generating the substrates ATP and pyruvate for anaerobic and
aerobic metabolism (1-6). This reaction is essentially irreversible
in vivo (Keq = 103 to 104) and appears to be one of the major
control points for the regulation of the glycolytic flux. In
addition, both substrate and products of this reaction feed into
a number of energetic and biosynthetic pathways, placing PK at a
primary metabolic intersection. The X-ray crystal structures of
several PKs from different species (e.g., Escherichia coli, Leish-
mania mexicana, Bacillus stearothermophilus, cat, rabbit muscle,
human erythrocyte, and yeast) revealed a high degree of struc-
tural homology in the general PK topology (2-5, 7). PKs exist as
homotetramers of identical subunits of ∼50-60 KDa depending
on species, each consisting of three to four domains: A, B, C, and
N-terminal domains (Figure 1). The N-terminal helical domain is
†This work was supported by funding from Genome Canada,
Genome British Columbia, Vancouver General Hospital and UBC
Hospital Foundation, and SARS Accelerated Vaccine Initiative to the
PRoteomics for Emerging PAthogen REsponse (PREPARE) Project.
Computer equipment in PREPARE Project’s Computational Genomics
research was also supported by in-kind contribution from IBM Health-
care and Life Sciences.
*Corresponding author. E-mail: ethan@interchange.ubc.ca. Tel: 604-
875-4011. Fax: 604-875-5654.
1Abbreviations: S. aureus, Staphylococcus aureus; MRSA, methicillin-
resistant S. aureus; PK, pyruvate kinase; CT, extra C-terminal domain;
PEP, phosphoenolpyruvate; R5P, ribose 5-phosphate; FBP, fructose
1,6-bisphosphate; AMP, adenosine 50-monophosphate; ADP, adeno-
sine diphosphate; PFK, 6-phosphofructokinase; F26BP, fructose 2,6-
diphosphate; HPr, phosphoryl carrier protein; PPDK, orthophosphate
dikinase; PTS, phosphotransferase system; EI, enzyme I.
r
2010 American Chemical Society
Published on Web 07/30/2010
pubs.acs.org/Biochemistry

7734 Biochemistry, Vol. 49, No. 35, 2010
Zoraghi et al.
F
IGURE 1: Amino acid sequence alignment of MRSA PK with pyruvate kinases from other species. Accession numbers: MRSA, (this study;
YP_041163.1); B.ste, B. stearothermophilus (BAA02406); E.PK1, E. coli PK1 (NP_288110.1); E.PK2, E. coli PK2 (ACT72239); S.typ,
S. typhimurium (NP_460343.1); M.tub, M. tuberculosis (NP_216133.1); C.tra, C. trachomatis (NP_219839.1); hM1, human M1 (NP_
872270.1); hM2, human muscle isozyme M2 (NP_002645.3); hLR1, human LR1 (NP_000289.1); hLR2, human LR2 (NP_870986.1); D.mel,
D. melanogaster(NP_732723.1). The 3D domains (N/A/B/C/CT) are indicated byverticallines. BoldAAs indicate PK signature sequences. Thick
cursive letters A, P, and D indicate ATP, PEP, and divalent cation binding sites, respectively. The binding sites of the sugar, 1-phosphate, and
6-phosphate moieties of FBP are indicated by closed circles, open squares, and closed squares, respectively. Numbers 1 and 2 indicate deletions in
the MRSA sequence compared to the host homologues. According to the percent conservation, columns of residues having 100% conservation
are shaded.
absent in prokaryotic PKs and can be removed from human
erythrocyte PK with no effect on its stability or activity (8). The A
domain with β/R-barrel topology is located between the B
(with β-barrel structure) and C (with R/β topology) domains. The
active site lies at the interface of the A and B domains in each of
the four subunits while the binding site for allosteric effector
appears to be located at the C domain (Figure 1) (2, 3).
While there are four mammalian PK isozymes, M1, M2, L
(liver), and R (red blood cell), with different primary structures,
kinetic properties, and tissue distributions to satisfy the metabolic

Article
Biochemistry, Vol. 49, No. 35, 2010 7735
requirements of various tissues, most bacteria and lower eukar-
yotes have only one PK isoenzyme; only a few bacterial species
(i.e., E. coli and Salmonella typhimurium) have two isoenzymes.
PK is subject to various types of regulation, such as tissue-
specific isozyme distribution, phosphorylation-dephosphoryla-
tion, and allosteric regulation, to regulate the switching between
glycolysis and gluconeogenesis (9). Allosteric regulation, in
particular, provides mechanisms for tight modulation of PK
enzymes. All known PKs (except mammalian muscle M1 iso-
form) exhibit an inactive/active reversible association depending
on allosteric activation by PEP and one or more allosteric
effector(s) whose precise chemical nature depends on the source
of enzyme.
a conserved PEP utilizer superfamily domain as an extra C-termi-
nal (CT) sequence, the role of which is not yet understood
(6, 16, 28, 29). We characterized MRSA PK as the second CT-
containing PK studied to date. We also extensively defined the
contribution of the CT domain to the holoenzyme structure,
function, and mode of regulation by studying the kinetic proper-
ties, regulatory behavior, and biophysical properties of a trun-
cated MRSA PK mutant in which the CT domain was deleted.
The results reported below improve our understanding of the
control mechanisms governing primary carbon metabolism in
S. aureus. They also provide insights into the functional features
and mechanisms of regulation of S. aureus PK and its significance
as a potential anti-MRSA drug target.
Fructose 1,6-bisphosphate (FBP) acts as a potent allosteric
activator of a number of PKs of different origin (e.g., mammalian
M2, L and R isozymes, yeast PK, and some bacterial PKs)
(1, 10, 11), whereas in trypanosomatid protozoans L. mexicana
and Chlamydia trachomatis the allosteric activator is fructose 2,6-
diphosphate (F26BP) (5, 12, 13). The relationship of structure to
this allosteric regulation of PK activity is not fully understood.
However, it was shown recently that the transition between
inactive T-state and active R-state involved a symmetrical 6ꢀ
rigid-body rocking motion of the A- and C-domain cores in each
of the four subunits. This also involved the formation of eight
essential salt-bridge locks across the C-C interface that provided
tetramer rigidity and a 7-fold increase in enzyme activity (14).
PKs have been isolated and characterized from numerous
bacterial species (e.g., E. coli (15), B. stearothermophilus (16),
Pseudomonas citronellolis (17), Streptococcus lactis (18), Lacto-
bacillus bulgaricus (19), Mycobacterium smegmatis (20), and
C. trachomatis (13). However, due to their general instability,
bacterial enzymes have not been extensively studied. We know,
nevertheless, that similar to eukaryotic PKs bacterial PKs
demonstrate tight allosteric control by a variety of metabolite
effectors. In E. coli, PK1 is inducible and activated by FBP,
whereas the enzymatic activity of PK2 is constitutive and
activated by AMP and ribose 5-phosphate (R5P) (21). The latter
may play an essential role in the production of ATP under
anaerobic conditions.
PKs has recently been drawing a lot of attention as a novel
target for antitrypanosomal, antileishmania, antimalaria, and
anticancer drugs (22-26). This has particular relevance, since the
high prevalence of multidrug-resistant organisms such as methi-
cillin-resistant Staphylococcus aureus (MRSA) has generated an
intensive search for alternative targets for development of new
antimicrobials. Potential target genes should be essential for
bacterial viability and pathogenesis (27). Until now, no studies of
this crucial metabolic enzyme have been carried out for S. aureus,
leaving its structural, kinetic, and regulatory properties un-
known. The current series of experiments were designed to
address these deficiencies. Our results show that S. aureus PK
carries out an absolutely essential function for survival of this key
Gram-positive pathogen. Moreover, we found that PK enzy-
matic activity increases during the exponential phase of S. aureus
growth. These data, together with the high level sequence and
likely structural divergence between bacterial and mammalian
PKs, indicate that PK is a potential novel antibacterial target.
Consequently, we expressed S. aureus PK as an active recombi-
nant enzyme and, for the first time, report a comprehensive
functional, structural, and biochemical characterization of the
MRSA PK protein. PKs from various species of staphylococci
and several other bacteria (e.g., Bacillus sp. and Listeria sp.) have
MATERIALS AND METHODS
Bacterial Strains and Plasmids. Epidemic methicillin-re-
sistant S. aureus (MRSA) strain sequenced at Sanger Centre,
U.K. (Sanger 252 NRS71), and S. aureus RN4220 (NCTC8325
NRS144) were obtained from NARSA (The network on anti-
microbial resistance in S. aureus). The staphylococcal plasmid
pNL9162 used for PK essentiality evaluation was from Sigma-
Aldrich.
S. aureus Pyk Gene Inactivation Using Targetron Tech-
nology. PK gene (pyk 1755 bp) was disrupted independently at
three different target sites (312, 357, and 390 bp) by insertion of a
group II intron in the sense orientation relative to pyk transcrip-
tion using targetron technology. Attempts were also made to
disrupt PK gene by insertion of the intron in the antisense orienta-
tion (77 bp). The pyk-targetron target fragments of 350 bp were
amplified by three specific primers (PK-IBS, PK-EBS1d, and
PK-EBS2) for each construct, according to the protocol of the
Targetron gene knockout system kit (Sigma-Aldrich, St. Louis,
MO) to retarget the RNA portion of the intron. Primers were
designed to introduce nucleotide substitutions into the IBS,
EBS2, and EBS1d regions of the LtrBL1 intron originating from
Lactococcus lactis using the InGex Intron Prediction Program
accompanying the targetron products (www.Sigma-Aldrich.
com/Targetronaccess). S. aureus intron-donor vector pNL9162
was used as a template for PCR reactions to construct pyk-
specific targetron donor plasmids pPK312s, pPK357s, pPK390s,
and pPK77a containing the pyk-specific IBS-EBS fragments
(350 bp) between the HindIII and BsrGI sites inserting LtrBLl
targetrons into the selected sites in the pyk gene. Pyk-specific
targetron donor plasmids were electroporated into S. aureus
RN4220. Transformants were grown until early log phase when
intron invasion was induced by 10 μM CdCl2 at 37 ꢀC. After
overnight induction, cells were isolated on BHI plates containing
10 μg/mL erythromycin. The intron insertions were identified by
colony PCR, using the genomic DNA from these mutants as
templates and specific PK primers flanking the predicted inser-
tion sites in the pyk gene. The amplified fragments were cloned
into pUC19 (Sigma-Aldrich) and sequenced to confirm the
intron insertion. Attempts to cure the pyk targetron plasmids
by plate-streaking the insertion mutants on BHI (no erythromycin)
at 37-40 ꢀC to identify erythromycin-sensitive mutants failed.
Due to the temperature sensitivity of the IEP-assisted splicing
reaction in targetron system, wild-type cells, transformants
having uninduced pyk donor plasmids, and pyk disruptants in
sense orientation were streaked on plates incubated separately at
32 and 43 ꢀC to access the essentiality of PK for growth. The
plates were incubated for 24-48 h and photographed. Control

7736 Biochemistry, Vol. 49, No. 35, 2010
Zoraghi et al.
parent strain and induced transformants of pPK390s were grown
in BHI medium overnight at 32 ꢀC, then diluted 1:20 into fresh
medium, and incubated for 4 h at 32 ꢀC or for 2 h at 43 ꢀC. RNA
was extracted and analyzed by RT-PCR, using PK-specific
primers flanking the pyk390s intron inserted in the pyk gene.
Primers sequences are available upon request. Control RT-PCR
experiments were performed when the initial reverse transcrip-
tion step was omitted or the reactions were carried out in the
absence of RNA substrate. Disruption of hsa, a previously con-
firmed essential gene in S. aureus, was performed as a control (30).
The 6-phosphofructokinase gene (pfk) was also disrupted
independently by insertion of the group II intron at position
363 in the antisense strand of the gene.
Evaluation of PK Expression and Enzymatic Activity
during the S. aureus Life Cycle. Turbidity growth curves were
generated for S. aureus strain RN4220 in BHI at 37 ꢀC by
measuring cell density at OD600. Cultures were inoculated with
bacterial suspensions at an initial OD600 of 0.1, equal to the con-
centration of approximately 107 CFU/mL. Growth rates were
determined for up to 24 h in three independent experiments
performed in duplicate. PK expression and enzymatic activity
were also determined at different time points of parallel cultures.
Cells were harvested at various time points, washed with PBS,
and then lysed for 30 min at 37 ꢀC in lysis buffer (20 mM Tris-
HCl, pH 7.5, 150 mM NaCl, 100 μg/mL lysozyme, 100 μg/mL
lysostaphin, 0.04% Triton X-100, 16 μg/mL DNase I, 1.6 mM
MgCl2, 0.5% NP-40, 1 mM DTT, and Complete protease
inhibitor). Cell debris was pelleted by centrifugation at 25000g
for 20 min, and soluble cell extracts were further filtered through
a 0.8 μm filter. MRSA cell extract (50 μg) was used to determine
PK enzymatic activity at different phases of S. aureus growth as
described above.
To assess PK protein expression, 2 μg of cell extracts obtained
from different time points was subjected to 12% SDS-PAGE
and transferred to polyvinylidene difluoride membranes (Millipore)
for immunoblotting. The primary antibody was a rabbit poly-
clonal anti-MRSA PK generated in this study, and the secondary
antibody was goat anti-rabbit horseradish peroxidase (BioSource
International). The blots were developed using an ECL chemi-
luminescence kit (Amersham Biosciences) and exposed to X-ray
film for 2-10 s, conditions under which each exposure to the film
does not reach saturation. Resultant films were scanned and
quantified using ImageJ software (NIH). The quantified protein
bands were plotted using the intensity of the band in the peak
fraction as 1 and quantifying the amount of the protein in
surrounding fractions as a percentage of that in the peak lane.
Blots were stained with Ponceau S after development to provide a
control for equal loading of the MRSA lysate.
Generation of PK Constructs. Genomic DNA of MRSA
strain Sanger 252 extracted using the Dneasy tissue kit (Qiagen)
was used as a template to generate the His-tagged MRSA PK
apoenzyme (Met1-Leu585) (MRSA PKWT), as well as a His-
tagged PK mutant truncated at the extra C-terminal domain
(Met1-Val472) (MRSA PKCT) by introduction of a stop codon at
the appropriate loci. The boundary for generation of the
C-terminal truncation was selected based on homology with other
extra C-terminal-containing PKs (i.e., B. strearothermophilus).
The following primer sets were used, creating appropriate
restriction sites (NdeI and XhoI sites underlined) for cloning of
MRSA PKWT (M27F, 50-CTACATATGAGAAAAACTAAA-
ATTGTATG-30, and M27R, 50-GTTCTCGAGTTATAGTAC-
GTTTGCATATCCTTC-30) and MRSA PKCT (M27F and
M27CTR, 50-GTTCTCGAGTTAAACTAGGTGGATTTTC-
ATC-30). The resulting PCR fragment for each construct was
cloned into NdeI and XhoI sites of the bacterial expression vector
pET-28a(þ) (Novagen) providing an N-terminal 6ꢀ His tag to
generate pET-28a-MRSA PKWT and pET-28a-MRSA PKCT
.
DNA of E. coli strain O157:H7 (Sakai) (courtesy of Dr.
B. Finlay, University of British Columbia, Canada) was used as a
template to generate the full-length E. coli PK1 (NP_310410) and
PK2 (NP_310591) enzymes. The following primers were used for
cloning using NdeI/BamHI sites (underlined) for PK1, 50-CTA-
CATATGAAAAAGACCAAAATTGTTTGCACCATC-30 and
50-GTTGGATCCTTACAGGACGTGAACAGATGCGGTG-
TTAG-30, and NdeI/XhoI sites (underlined) for PK2, 50-CTA-
CATATGTCCAGAAGGCTTCGC-30 and 50-GTTCTC GAG-
TTACTCTACCGTTAAAATACGCGTGG-30. The resulting
PCR fragments were cloned as indicated above into the corre-
sponding sites of pET-28a(þ) to generate pET-28a-PK1 and
pET-28a-PK2 that generated N-terminally His-tagged recombi-
nant E. coli PK isoforms. The sequence and the correct reading
frame of all constructs were verified by sequencing.
Production of Anti-MRSA PK Antibody. Polyclonal PK
antibody was produced by immunization of rabbit with purified
recombinant His-tagged MRSA PKWT according to standard
protocols (Pacific Immunology Crop, Canada).
Expression and Purification of Recombinant His-Tagged
MRSA and E. coli PKs. MRSA and E. coli constructs in
pET-28a(þ) vector were used to generate relevant recombinant
PK proteins in E. coli BL-21(DE3). The proteins were expressed
and purified using Ni-NTA agarose (Qiagen) according to the
manufacturer’s protocol. Briefly, cells were grown to an absor-
bance of 0.4-0.5 at 600 nm in 2 ꢀ YT medium and then induced
with 0.1 mM IPTG for 3 h at 20 ꢀC. Cells were lysed by sonication
on ice (3 ꢀ 10 s bursts with a 30 s recovery between bursts) in lysis
buffer (0.2 mg/mL lysozyme, 50 mM Tris, pH 7.5, 10 mM MgCl2,
200 mM NaCl, 100 mM KCl, 10% glycerol, 10 mM imidazole,
0.5% NP-40, and 1 mM DTT containing Complete protease
inhibitor). Cell lysates were cleared by centrifugation (20000 rpm
in a Beckman JA-20 rotor) for 20 min at 4 ꢀC, and PK isoforms
were purified by batch binding to Ni-NTA resin. The resins were
then packed in columns (1 ꢀ 2 cm) and washed with 400 column
volumes of lysis buffer containing 30 mM imidazole. His-tagged
PK isoforms were eluted with the same buffer containing 300 mM
imidazole. The proteins were dialyzed overnight at 4 ꢀC versus
2000 volumes of ice-cold 30 mM Tris, pH 7.5, 25 mM KCl, 5 mM
MgCl2, 10% glycerol, and 1 mM DTT to remove imidazole. All
purification steps were done at 4 ꢀC; enzymes were flash-frozen
and stored at -70 ꢀC where activity was found to be stable for at
least 10 months. No loss of enzyme activity was observed with up
to five freeze-thaw cycles. The purity and physical integrity of
proteins were assessed using SDS-polyacrylamide gel electro-
phoresis (SDS-PAGE) followed by Coomassie Blue staining or
immunoblotting. Protein concentration was estimated by Brad-
ford assay (Bio-Rad protein assay) using bovine serum albumin
as standard.
Quaternary Structure Determination. The molecular masses
of the recombinant proteins were determined by size exclusion
chromatography. Purified His-tagged full-length and extra
C-terminally truncated MRSA PK (0.25 mg) in buffer B (30
mM Tris-HCl, 25 mM KCl, 3% glycerol, and 1 mM DTT) were
injected independently onto a Superdex 200 HR (10 mm ꢀ 30 cm)
column run on the AKTA Purifier liquid chromatography
system (Amersham-Pharmacia Biotech) equilibrated with the

Article
Biochemistry, Vol. 49, No. 35, 2010 7737
same buffer. The proteins were eluted at a flow rate of
0.7 mL/min at 4 ꢀC. Elution positions were determined using
the PK assay. For reference, 2.0 mg of a protein standard
mixture (Bio-Rad) was applied to the same column and eluted
using the same protocol. The molecular mass of each protein
was determined from the standard curve based on elution
volumes.
Measurement of Pyruvate Kinase Activity. PK activity
was determined using a modification of published methods (23),
by a continuous assay coupled to lactate dehydrogenase (LDH)
in which the change in absorbance at 340 nm owing to oxidation
of NADH was measured using a Benchmark Plus microplate
spectrophotometer (Bio-Rad). The reaction contained 60 mM
Naþ-HEPES, pH 7.5, 5% glycerol, 67 mM KCl, 6.7 mM MgCl2,
0.24 mM NADH, and 5.5 units of L-LDH from rabbit muscle
(Sigma-Aldrich) in a total volume of 200 μL. Two millimolar
ADP and 0-30 mM PEP (for experiments to determine S0.5 for
PEP in the presence or absence of 1 mM AMP) or 0-5 mM ADP
and 20 mM PEP (for experiments to detrmine S0.5 for ADP) or
2 mM ADP and 10 mM PEP (for experiments to locate the elution
position of proteins or assess specific activity of native enzyme in
different phases of S. aureus growth) were added as substrates.
Reactions were initiated by addition of one of the following PK
enzymes: MRSA PKWT (15 nM), E. coli PK isoforms (15 nM),
MRSA PKCT (200 nM), or MRSA cell lysate (50 μg). PK activity
proportional to the rate of change at 340 nm was expressed as
specific activity (μmol min-1 mg-1), which is defined as the
amount of PK that catalyzes the formation of 1 μmol of either
product/min. The apparent S0.5, Hill coefficient (h), and Vmax or
kcat values were determined by allosteric sigmoidal nonlinear
regression analysis of data using the Prism software package of
Graph Pad Inc. (San Diego, CA). The molecular masses of the
MRSA His-tagged wild-type (PKWT), truncated (PKCT), and the
native PK enzyme as monomers are assumed to be 65168, 53639,
and 63142 Da, respectively. In all studies, less than 10% of total
PEP was exhausted during the reaction. Reactions were per-
formed at 30 ꢀC for 5 min. All values determined represent three
measurements, each in triplicate.
F
IGURE 2: Phylogenetic (neighbor-joining) tree constructed using
pyruvate kinase amino acid sequences. The accession numbers, in
addition to those indicated in Figure 1, are as follows: Saccaromyces
cerevisiae(NP_009362.1), Bacillussubtilis(NP_390796), Haemophilus
influenzae (AAX88293), and Leishmania infantum (XP_001468820).
Sequences were aligned using the ClustalW software (36). The phylo-
genetic tree was constructed using the phylML (59).
RESULTS
Bioinformatic Analysis of MRSA PK. Genome sequence
analysis of a number of S. aureus strains for which such sequences
are available (e.g., MRSA252: NC_002952.2) revealed that pyk in
S. aureus exists as a single copy gene located 22 nucleotides
downstream of 6-phosphofructose (PFK) gene (pfk) in a tandem
pfk-pyk arrangement. This suggests that pyk and pfk genes form
a bicistronic operon in S. aureus. No significant ORF was found
in the 345 nucleotides downstream of the pyk gene, indicating
that the tandem pfk-pyk arrangement was not associated with
another gene. The operon association between pyk and pfk was
previously observed in some other Gram-positive organisms such
as L. bulgaricus (31, 32), L. lactis (33), Mycoplasma genitalium
(34), B. stearothermophilus, Streptococcus bovis, Bacillus psychro-
philus, and Bacillus licheniformis (35). Although both of these
enzymes are closely related in glycolysis, the biological signifi-
cance of this operon remains unclear (28).
The amino acid sequence of the MRSA PK was compared to
the sequences present in the GenBank/EMBL databases using
BLAST (36). As expected, high homologies between bacterial
PKs were found in the protein databases. The highest score was
obtained for Staphylococcus sp. (85-100% identity). The next
highest homologies were with Bacillus sp. (e.g., 62% identity with
PK from B. stearothermophilus), Enterococcus faecalis (60%
identity), Listeria sp. (58% identity), E. coli PK1 (48% identity),
and E. coli PK2 (39% identity). The higher identity with the
type I isoenzyme of E. coli (e.g., PK1) suggests that the MRSA PK
belongs to the type I group. The phylogenetic relationships repre-
sented in Figure 2 showed a wide divergence between MRSA PK
and human isozymes. Phylogenetic analysis also clearly revealed
the significant evolutionary distance existing between the type I
and type II (e.g., E. coli PK2) isoenzymes in Gram-negative
bacteria. The distinction, however, is not so definite in Gram-
positive microorganisms. PK from B. stearothermophilus (16),
which is known to have all of the characteristics of a type II iso-
enzyme, is found clustered with the other Gram-positive enzymes.
Thus, the appurtenance of the PK from a Gram-positive bacte-
rium to one type of E. coli isoenzyme cannot be determined solely
Definition of Major Kinetic Parameters. Turnover num-
ber (kcat) is the number of catalytic events per second per mole of
active site. S0.5 is the substrate concentration at which the
reaction velocity is half-maximal. Hill coefficient (h) is an
empirical parameter related to the degree of cooperativity; values
larger than unity indicate positive cooperativity among ligand
binding sites of different subunits.
Calculation of Free Energy of Binding. The value for the
Gibbs free energy change, ΔG, that occurs by association of PEP
or ADP with MRSA PK is related to the equilibrium association
constant for the interaction and was calculated using eq 1:
ΔG ¼ - RT ln K
ð1Þ
where K = Km, R is the ideal gas constant (equal to 1.98 ꢀ 10-3
kcal deg-1 mol-1), and T is the temperature at which the assay
was done (303 ꢀK).
The contribution of CT domain to the Gibbs free energy of bind-
ing in an enzyme-transition state complex was calculated from
eq 2:
ΔΔG ¼ ΔGWT - ΔGCT
ð2Þ
ΔΔG is the change in free energy of binding in enzyme-transition
state complexes attributable to the CT truncation.

7738 Biochemistry, Vol. 49, No. 35, 2010
Zoraghi et al.
on the basis of primary sequence homologies; biochemical
properties must also be considered.
of bacteria including S. aureus (38). In order to formally prove the
essential nature of this protein, the targetron system was used to
knockout PK gene with an intron donor plasmid expressing
LtrBLl and LtrA under the control of Pcad promoter with a
temperature-sensitive replicon (30). Toward this end, pyk was
disrupted independently at one target site in antisense orientation
(pyk77a) and three different target sites in sense orientation
(pyk312s, pyk357s, and pyk390s). More than 200 transformants
were obtained from the electroporation of strain RN4220 with
each pyk targetron donor plasmid that mediated intron integra-
tion into the site-specific pyk antisense and sense strands (e.g.,
pPK77a, pPK312s, pPK357s, and pPK390s, numbered from its
translation start codon).
Cadmium induction was necessary to obtain pyk disruptants
since Pcad promoter had a low basal level of transcription in the
absence of cadmium. Ten colonies were PCR-screened from cells
without cadmium induction for each pyk targetron, and none of
them had the desired disruption. In contrast, after cadmium
induction, 4, 6, and 9 of 10 colonies had the desired disruption for
pyk312s, pyk357s, and pyk390s targetrons, respectively. Thus the
total insertion frequency of the intron at nt 312|313s, 357|358s,
and 390|391s of pyk was calculated to be (approximately) 40%,
60%, and 90%, respectively. Attempts failed to get any pyk
disruptant with pyk77a despite screening up to 100 induced
colonies. The inability to get pyk intron insertion in the antisense
orientation may indicate the essentiality of the pyk gene, since in
principle, targetron inserted in the antisense orientation is
transcribed into the complement of the intron RNA sequence
and cannot be spliced, yielding an unconditional pyk disruption.
PK gene fragments were amplified from a subset of induced
pyk390s targetrons (Figure 3A, lanes 1-7; 1.2 kb) versus the
control parent cells (Figure 3A, WT; 0.28 kb) and uninduced
pPK390 transformant (Figure 3A, T; 0.28 kb). The size and
sequencing results (data not shown) of the amplified pyk-intron
fragments verified the presence of the LtrBL1 intron integrated
into the site-specific pyk sense strand (e.g., at the site between nt
390 and 391 of pyk). The smaller PCR product (Figure 3A, WT
and T; 0.26 kb) was derived from pyk gene without the inserted
intron, and the larger product (Figure 3A, lanes 1-7; 1.2 kb) is
derived from the pyk gene containing the inserted intron.
LtrA protein could potentially remobilize or splice out the
inserted intron if it was integrated in the sense orientation;
therefore, it was desirable to cure the targetron plasmids from
the disruptants prior to analysis. However, the donor plasmid
could not be cured if the targeted gene was essential because the
IEP-assisted splicing of the inserted intron was required to
produce essential protein. Several attempts made to cure the
pyk targetron plasmids (pyk312s, pyk357s, and pyk390s) were
unsuccessful, indicating the essential nature of the pyk gene.
Nevertheless, temperature sensitivity of the IEP-assisted splicing
reaction in the targetron system made it possible to obtain
conditional disruptants for introns integrated in the sense ori-
entation by growth at elevated temperature (e.g., 43 ꢀC). PK gene
disruption by targetron yielded conditional pyk disruptants that
grew at 32 ꢀC (inserted intron spliced out) but not at 43 ꢀC
(inserted intron disrupted pyk) for pyk390s (Figure 3B), pyk312s,
and pyk357s (data not shown). The hsa24s targetron was also
used as a control to disrupt the previously shown essential hsa
gene (Figure 3B). To confirm that the temperature sensitivity of
the pyk disruptants was due to the temperature-sensitive IEP-
assisted splicing reaction of the inserted intron, the splicing of the
pyk390s intron inserted in the PK gene was assayed after the
MRSA PK was found to have an extra C-terminal extension
(CT) of 112 residues reported previously only in Gram-positive
bacteria such as Bacillus sp. (28, 35), L. bulgaricus (31), and
L. lactis (31) (Figure 1). The MRSA CT domain composed of
residues Gly473-Leu585 (Figure 1) encodes a motif (PROSITE
no. PS00742) characteristic of mobile PEP-utilizing enzymes such
as pyruvate, orthophosphate dikinase (PPDK, EC 2.7.9.1) found
in plants, PEP synthase (EC 2.7.9.2) essential for gluconeogen-
esis, or the PEP-protein phosphotransferase (EC 2.7.3.9), i.e.,
enzyme I (EI) of the PEP-dependent sugar phosphotransferase
system (PTS). In these enzymes, the phosphoryl group trans-
ferred to PEP is transiently carried by a conserved histidine
residue. Interestingly, this histidine residue was found to be
replaced by a proline in S. aureus PK (e.g., P; residue 539 in
MRSA252). The biological role of this domain in PKs has not yet
been fully elucidated. However, it has been suggested that the CT
extension may play a role in the structural stability of the enzyme
through its interaction with the A domain at the subunit inter-
face (6). The presence of this extra C-terminal domain suggested a
possible dual function for the MRSA PK, where PEP can be
transformed into pyruvate either by the classical PK activity that
produces ATP or by the potential phosphotransferase activity
that does not produce ATP but possibly phosphorylates another
target that could be either a small metabolite or a protein [e.g., the
phosphoryl carrier protein (HPr) of the PTS]. In the present
study, we analyzed the actual impact of CT domain on MRSA
PK structure and function by characterization of a C-terminally
truncated PK mutant.
The sequence alignment of PKs from various eukaryotes and a
wide variety of both Gram-positive and Gram-negative bacteria
using ClustalW software (36) revealed sequence divergence and
several amino acid insertions and deletions which clearly deline-
ate eukaryotic PKs from those of bacterial origin (Figure 1). The
homologies of the MRSA PK amino acid sequence to the
corresponding sequences of human M1, M2, L, and R were 45%,
44%, 43%, and 43%, respectively, due to the highly conserved
residues within PK. Multiple alignment of amino acid sequences
of bacterial PKs with those of human enzymes (Figure 1) revealed
that the known binding sites for ADP, PEP, and cations, located
in the A and B domains, are well conserved (76-100%), whereas
sites for effector binding, located in domain C, are not conserved.
In addition, some deletions in the A domain and in the vicinity of
the effector binding site in the C domain were found to be unique
to bacterial PKs (Figure 1). These unique sites could be targets
for the design of selective inhibitors. In this regard, homology
modeling (Cherkasov et al., unpublished results) showed that the
high level of sequence (Figure 1) and structural divergence
between bacterial and mammalian PKs should permit the devel-
opment of specific inhibitors for bacterial PK.
Essentiality Testing of S. aureus PK. As part of a genome-
scale analysis of essentiality in S. aureus using inducible antisense
RNA expression, Forsyth and colleagues (37) identified that PK
is critical for growth. However, it was not clear whether expres-
sion of PK antisense RNA leads to lethal isogenic or growth-
defective strains. Moreover, definitive determination of PK
essentiality using antisense inhibition is particularly problematic
due to the potential polarity effect of PK-specific antisense RNA
on the expression of the upstream pfk. Recently, “targetron”, a
gene knockout system that utilizes the L. lactis Ll.LtrB group II
intron, has been adapted for gene disruption in a growing number

Article
Biochemistry, Vol. 49, No. 35, 2010 7739
F
IGURE 3: Disruption of the PK and PFK genes in S. aureus using targetron-mediated intron insertion. (A) Detection of pyk390S-intron
integration. Colony PCR was performed on control parent strain (RN4220:WT) and pyk disruptants (1-7) and uninduced pPK390 transformant
(T) utilizing specific PK primers for the DNA flanking intron insertion site (390|391s). Cells were grown at 32 ꢀC. (B) Plating assay. Wild-type
RN4220 (WT), induced pyk disruptants obtained with pPK390 (1-6), and hsa disruptant obtained from pNL9164 were streaked on BHI agar
plates at 32 and 43 ꢀC. The plates were incubated for 24-48 h and photographed. (C) RT-PCR assay of pyk390-intron splicing from the pyk gene.
Control parent strain (RN4220:WT) and induced transformants of pPK390 (1-2) were grown in BHI medium overnight at 32 ꢀC, then diluted
1:20 into fresh medium, and incubatedfor 4 h at 32ꢀC orfor 2 h at43 ꢀC. RNA was extracted and analyzedby RT-PCR, using PK-specific primers
flanking the pyk390 intron inserted in the pyk gene. Controls showed no products if the initial reverse transcription step was omitted, indicating
that the amplified bands in the experimental samples are derived from cellular RNA. (D) Detection of pfk363A-intron integration. Colony PCR
was performed on control parent strain (RN4220:WT) and pfk disruptants (1-4) and uninduced pPFK363 transformant (T) utilizing specific
PFK primers for the DNA flanking intron insertion site (363|364a). Cells were grown at 32 ꢀC. (E) Plating assay. Wild-type RN4220 (WT), cured
uninduced pPFK363 transformant (T), cured inducedpfk disruptants obtainedwithpPFK363 (1-3), and hsa disruptantobtainedfrompNL9164
were streaked on BHI agar plates at 32 and 43 ꢀC. The plates were incubated for 24 h and photographed.
growth shift to 43 ꢀC by RT-PCR using PK-specific primers
flanking the intron-insertion site (Figure 3C). At 32 ꢀC, in the wild-
type (WT) strain and the uninduced transformant, the product
was a 0.26 kb band corresponding to PK mRNA (Figure 3C, WT
and T), and this same band, corresponding to mRNA with
spliced exons, was also the product in the pyk390s disruptant
(Figure 3C, 1). Strikingly, when cells were grown overnight at
32 ꢀC and shifted to 43 ꢀC for only a short time (e.g., 2 h), the wild-
type strain showed a single band (0.28 kb) corresponding to pyk
mRNA without the inserted intron as expected (Figure 3C, WT),
while the pyk disruptant showed substantial accumulation of the
larger band (1.2 kb) derived from the precursor RNA containing
the inserted intron, with concomitant reduction in the intensity
of the band corresponding to the mRNA with spliced exons
(Figure 3C, 1).
nonessential gene in S. aureus, as a nonconditional knockdown
strain that was generated using confirmed intron insertion to the
pfk at position 363 of the antisense strand grew even after curing
the donor plasmid at both 32 and 43 ꢀC (Figure 3D,E). These
results confirm the suggestion that PK is essential in S. aureus but
contradict the results of a previous work demonstrating PFK
essentiality using an antisense strategy (37).
PK Expression and Enzymatic Activity Increases during
Exponential Phase of S. aureus Growth. It was previously
shown that almost complete depletion of glucose occurs during
the exponential phase of S. aureus growth (39). We expected that
glucose depletion would correlate with increased PK expression
and/or activity in the exponential phase of growth so that
inactivation of essential PK protein could lead to growth inhibi-
tion by limiting the ability of S. aureus to catabolize glucose. In
order to test this hypothesis and characterize the role of the PK in
the S. aureus life cycle, PK expression and specific enzymatic
activity was analyzed during growth of S. aureus in liquid culture
(Figure 4). Expression of PK protein was found to be increased
In summary, results of pyk targetron inactivation at four
independent different target sites in both sense and antisense
strands indicated that the PK gene is an absolutely essential gene
for S. aureus viability. On the other hand, PFK was identified as a

7740 Biochemistry, Vol. 49, No. 35, 2010
Zoraghi et al.
F
IGURE 4: PK expression and enzymatic activity profile during S. aureus life cycle. Two independent S. aureus cultures were grown in parallel in
BHI after inoculation with an initial cell density of OD600 = 0.1. (A) At indicated time points, an aliquot was removed, appropriate dilutions were
made, and the cell density (b) was determined at OD600. Aliquots were also removed at the exponential, postexponential, and stationary phases of
growth (1-5, 7-8, and 24 h, respectively), and specific PK activity was determined (0) as described in Materials and Methods. (B) Expression of
PK was detected using anti-PK polyclonal antibody and quantified as described in Materials and Methods. The same gel was stained with
Coomassie Blue to show equal protein loading (right panel). The results presented are representative of three independent experiments.
(by 50%) during the exponential phase of growth (e.g., 1-4 h),
reaching maximal levels as bacteria entered the postexponential
phase of growth (e.g., 5-6 h) (Figure 4B). PK enzymatic activity
was also found to rapidly increase (by 2.8-fold) during the expo-
nential growth phase (e.g., 1-4 h), and this correlated with
expression of PK protein up until approximately 5 h. In contrast,
during the postexponential phase of growth, there was a sharp
drop in PK enzymatic activity while the PK protein levels remain
relatively constant, indicating posttranscriptional regulation of
PK activity in the stationary phase of growth. In addition, the
minimal PK activity in the stationary phase might be a result of
less substrate (PEP) availability due to the reduced pool of
glucose. These data show that PK expression and enzymatic
activity clearly correlate with the phase of bacterial growth and
are highly regulated during the bacterial life cycle (Figure 4A).
The important role of PK in S. aureus growth is in agreement
with the results of previous work demonstrating a substantial
reduction of growth rate (by 70%) in a PK mutant of B. subtilis (40).
Expression and Purification of PK Proteins. Functional
features of the MRSA PK were defined by creating and
characterizing the biophysical and biochemical properties of
recombinant His-tagged proteins including wild-type MRSA
PK containing the full-length sequence, PKWT (Met1-Leu585),
as well as a PK mutant truncated at the extra C-terminal domain
PKCT (Met1-Val472). Wild-type E. coli PK1 and PK2 isoforms
were used as controls for determination of enzymatic character-
istics of MRSA PKs. All proteins were expressed at high levels
(∼30-40 mg/L of culture) using the bacterial expression system.
Following purification as described in Materials and Methods,
each exhibited a high degree of purity (>98%) as determined by
SDS-PAGE [12% (w/v) gel] followed by Coomassie Brilliant
Blue staining (Figure 5) or Western blot analysis for MRSA PKs
using a polyclonal MRSA PK-specific antibody (results not shown).
The estimated molecular mass of each of the respective bands on
SDS-PAGE correlated well with that predicted based on the
amino acid composition for each PK protein (Figure 5), indicat-
ing that all PK constructs were expressed as expected full-length
proteins. MRSA PK proteins were further characterized with
respect to their quaternary structure, kinetics of catalytic activity,
and allosteric properties.
Assessment of the Oligomeric State of the PK Con-
structs. The molecular mass of wild-type PK was estimated by
gel filteration on a Superdex 200 HR column. The gel filtration
elution position of the MRSA PK was between those of the
standard proteins: thyroglobulin (MW = 670 kDa) and bovine
γ-globulin (MW = 158 kDa). The calculated molecular mass of
the wild-type MRSA PK appeared to be 250 kDa (Figure 5B).
This value was approximately quadruple that of the predicted
molecular mass of a monomer of this construct (65.1 kDa) based
on amino acid composition, suggesting that MRSA PK is
tetrameric. The eluted fractions corresponding to the tetrameric
form of PK were enzymatically active. Hence, like almost all
pyruvate kinases from other organisms, active MRSA PK exists
as a homotetramer. To investigate the impact of the CT domain
on the quaternary structure of the enzyme, PKCT, lacking the CT
domain, was also analyzed. Based on its elution position, the
calculated molecular mass was shown to be 215 kDa (Figure 5B)
for PKCT, consistent with a tetrameric structure compared with
that predicted from the amino acid composition (53.1 kDa).
Compared with wild-type PK, PKCT migrated as a somewhat
broader peak on gel filtration chromatography. This suggested
that the extra C-terminal domain of PK may contribute to
stabilizing the holoenzyme quaternary structure. The protein
concentration of the peak fractions for wild-type PK following
the Superdex 200 HR chromatography was determined by the
Bradford method to be 250 nM, indicating that the KD for

Article
Biochemistry, Vol. 49, No. 35, 2010 7741
F
IGURE 5: SDS-PAGE and size exclusion chromatography of recombinant wild-type and truncated MRSA PKs. (A) 5 μg aliquots of wild-type
(PKWT), C-terminally truncated mutant (PKCT), and E. coli PK1 and PK2 proteins purified to homogeneity through the Ni-NTA chromato-
graphy step as described in Materials and Methods were applied to lanes 1-4, respectively. Low molecular weight (LMW) standards (Pharmacia
Biotech) were applied as size markers. Proteins were visualized by staining the gel with Coomassie Brilliant Blue dye. (B) Wild type [PKWT (3), left
panel] and C-terminally truncated mutant [PKCT (O), right panel] were chromatographed separately on a 10/30 column of Superdex 200 HR, and
positions of the proteins emerging in the effluent were monitored by PK activity as described in Materials and Methods. The molecular mass of
each protein was determined from the standard curve generated from experiments with a protein standard mixture (Bio-Rad) applied to the same
column and eluted using the same protocol. Elution positions of bovine thyroglobulin (670 kDa) and bovineγ-globulin (158 kDa) are indicated as
separate curves (9). Results in these figures are representative of three experiments.
formation of a stable tetramer in MRSA PK is <250 nM. This
finding indicated that tetramerization of the PK enzyme is not
mediated by the CT domain. However, it does not rule out the
contribution of the CT domain to tetramer stability.
Kinetic Properties of the Recombinant Wild-Type and
Mutant MRSA PK. The specific activity (100 units/mg at
30 ꢀC) of purified MRSA PK is comparable to values reported
for other bacteria (13, 18-20, 41). To characterize the catalytic
function of purified MRSA PK and to examine the impact of
potential interactions of the CT domain on enzyme catalytic
function and affinity for substrate, steady-state kinetics of
recombinant (PKWT) and truncated (PKCT) MRSA PK were
determined with respect to PEP in the absence and presence of
1 mM AMP and with respect to ADP as described in Materials and
Methods (Figure 6). Parameters derived from these kinetics are
summarized in Table 1. In the absence of the activator (AMP),
both wild-type and truncated enzymes exhibited sigmoidal satu-
F
IGURE 6: Steady-state kinetics of wild-type and extra C-terminally
ration curves for PEP, with comparable cooperation as expressed
by Hill coefficients (h) of 2.8 ((0.4) and 2.3 ((0.5), respectively
(Figure 6A,B), suggesting homotropic allosteric activation of
enzyme by its substrate. These data indicate that the removal of
the CT domain has minimal effect on the substrate cooperative
interactions of enzyme. However, truncation of the CT domain
produced a significant decline in the PKCT catalytic activity (with
truncated MRSA PK. Steady-state kinetics of recombinant wild-type
(A) and truncated (B) PK in the absence (O) or presence (3) of 1 mM
AMP as a function of PEP. Kinetics of wild-type (C) and truncated
(D) MRSA PKs against ADP at fixed concentration of PEP (20
mM). PK assays were conducted as described in Materials and
Methods. The data are derived from one of three experiments
performed in triplicate and analyzed using Prism Graphpad software
(single site model). Error bars indicate range of values within the
single experiment shown.
kcat value less than 6.8% of that for PKWT) and a modest decline
in affinity for PEP (with S0.5 value of 1.6-fold that for PKWT
)
The presence of activator (AMP) shifted the dependence of the
wild-type initial velocity on the PEP concentration to a hyper-
bolic saturation curve, lowering h to 1.6 ((0.1), indicating
decreased cooperativity (Figure 6A) as has been described for
(Figure 6A,B and Table 1). These results were verified using four
separate preparations of purified PKCT and show for the first
time the importance of the CT domain for efficient PK catalysis.

7742 Biochemistry, Vol. 49, No. 35, 2010
Zoraghi et al.
PK from other sources (7, 32, 36). The presence of AMP led to
11-fold decrease in the S0.5 of PEP under nonsaturation condi-
tions. Similarly, the presence of activator in the kinetics toward
PEP shifted the sigmoidal curve of PKCT to a hyperbole, while
it had no effect on enzyme cooperativity (i.e., h = 2.9 ( 0.3)
(Figure 6B). Inspection of the data in Table 1 shows that the
apparent S0.5 (PEP) values of truncated protein (PKCT) were
increased with respect to those of wild-type protein both in the
absence (1.6-fold) and in the presence (3.6-fold) of 1 mM AMP.
Both proteins exhibited increased catalytic activity (kcat) upon
addition of AMP (Figure 6A,B and Table 1). However, the fold
increase for truncated PK was significantly higher than that of
the wild-type PK (kcat values for wild-type and truncated PKs
were 180.8 and 78.2 s-1, respectively). This significant increase
indicated that allosteric stimulation could restore the negative
influence of the extra C-terminal domain truncation on the basal
PK catalytic activity.
Steady-state kinetics of the wild-type PK and PKCT as a
function of ADP are shown in Figure 6C,D. Parameters derived
from these kinetics are summarized in Table 1. Both proteins
exhibited hyperbolic response to ADP with essentially identical
S0.5 values (0.4 ( 0.10) and comparable Hill coefficients (i.e., 1.1
and 0.8 for PKWT and PKCT, respectively) indicating no enzyme
cooperativity. However, PKCT had markedly reduced kcat value
(less than 4.9% that for PKWT), indicating that removal of the
CT domain also produced a particularly significant decline in the
PK catalytic activity toward ADP. The fact that the catalytic
activities of PKCT toward both PEP and ADP were profoundly
decreased compared to those of wild-type PK indicated that the
CT domain may contribute to stability of the tetramer.
The energy involved in interactions of PEP and ADP with
PKWT were quantified from the Km values for the respective
substrate by calculating the Gibbs free energy of binding (ΔG) of
3.0 and 4.7 kcal mol-1, respectively, as described in Materials and
Methods. These values reflected the affinities with which the
respective ligands bind to the PK catalytic site. The magnitude of
the contribution of the CT domain to the binding energy in
enzyme-transition state complexes (ΔΔG) was quantified by
calculating the Gibbs free energy of binding (ΔG) of PEP and
ADP in PKCT compared to the free energy of binding of these
ligands in PKWT. The change in the free energy of binding (ΔΔG)
of PKCT for PEP and ADP was equivalent to 0.28 and 0 kcal
mol-1, respectively, indicating that the free energy of binding in
the PKCT with PEP was slightly reduced when compared to that
of PKWT. The fact that the truncated mutant bound ADP with
essentially the same affinity as that of the wild-type PK indicated
that overall structures of the truncated protein were preserved
and that differences in its kinetic parameters were not due to
nonspecific conformational effects induced by truncation. These
data, on the whole, showed that there are no significant differ-
ences in the substrate affinity and allosteric regulation behavior
between wild-type enzyme and truncated mutant. The findings
suggest that the CT domain truncation only minimally perturbed
the substrate and effector binding sites. However, the profoundly
decreased catalytic activity of the CT domain-deleted mutant
suggested the essential role of this domain in activity and perhaps
stability of the MRSA PK in the active conformation.
Interestingly, neither wild-type nor CT truncated MRSA PK
proteins responded significantly to either FBP (1 mM) or glucose
6-phosphate (1 mM) over a wide range of PEP and ADP
concentrations, under both saturating and nonsaturating condi-
tions (data not shown).

Article
Biochemistry, Vol. 49, No. 35, 2010 7743
Table 2: Effectors of the MRSA PKa
fold activation
physiologic
concnb (mM)
activator
activator
at 10 mM
EC50
activator
at 1 mM
(mM)
h
MRSA PK
AMP
ATP
0.28
1.5
1.3c
15
4.1
1.9
4.6
1.0
0.8
1.7
0.3
4.5
0.0
4.6
0.3
0.0
0.4
0.0
0.014
NDe
0.005
ND
1.6
ND
1.2
R5P
FBP
ND
1.4
citrate
oxalate
Pid
2
2.7
ND
ND
ND
ND
1.2
0.37
MRSA PKCT
AMP
ATP
0.28
1.5
1.3c
15
58
1.1
143
1.1
0.9
3.4
0.2
32
0.05
139
0.5
0.065
ND
0.12
ND
1.3
3.3
ND
2.9
R5P
FBP
ND
ND
ND
1.2
F
IGURE 7: The allosteric regulation of MRSA PK. Kinetic response
citrate
oxalate
Pid
2
0.1
of recombinant wild-type (A) and truncated (B) MRSA PK as
compared to that of E. coli PK1 (C) and E. coli PK2 (D) to AMP
(O), R5-P (0), FBP (3), and ATP (4). Enzymatic activity was
assayed as described in Materials and Methods. The ADP and PEP
concentrations were fixed at 2 and 5 mM, respectively. Percent of
activity is based on the rate obtained in the absence of compounds.
ND
ND
3.0
ND
0.33
0.1
aRelative activities are presented. All values are the mean of three experi-
ments of triplicate determinations. bAccording to refs 42 and 43. cConcen-
tration of pentose phosphates denotes the combined pools of R5P, ribulose
5-phosphate, and xylose 5-phosphate. dInorganic phosphate. eND: cannot
be determined.
Allosteric Regulation Profile of MRSA PK. To map the
allosteric regulation profile of MRSA PK, specific activities of
wild-type and truncated MRSA PK (Figure 7A,B) were deter-
mined at varying concentrations of FBP, AMP, ribose 5-phos-
phate (R5P), ATP, citrate, oxalate, and inorganic phosphate (Pi).
Values of effects are reported in Table 2. The activation profile of
each PK by effectors was presented as fold activation compared
to that of the control (no compound) (Figure 7). Wild-type
MRSA PK was activated by AMP and R5P with half-maximal
concentrations (EC50) of 14 and 5 μM, respectively. PKCT was
also activated by AMP and R5P but at greater magnitude
compared to that of wild type. AMP and R5P at 1 mM showed
58- and 143-fold stimulatory effects on PKCT, respectively, while
they activated wild type by 4.1- and 4.6-fold, respectively, at
conditions tested (Table 2). Nevertheless, a higher concentration
of activator was needed for PKCT activation (EC50 values for
AMP and R5P were 4.8- and 24-fold larger than that of the wild-
type enzyme, respectively). This significant difference in magni-
of the enolpyruvate moiety of PEP and pyruvate) activated both
wild-type PK and PKCT at 1 mM. At higher concentration (e.g.,
10 mM), the stimulatory effect of oxalate was preserved for PKCT
,
while it was inhibitory for wild-type enzyme (60%) (Table 2).
Citrate inhibited enzyme activity of both PK proteins by 90% at
10 mM.
Interestingly, ATP exhibited a biphasic regulatory effect on
MRSA PK activity in the presence of excess of Mg2þ (6.7 mM).
Wild-type PK was activated by 2-fold at the physiologic range of
ATP (e.g., 1 mM), while it was completely inhibited at g2.5 mM
ATP in a noncompetitive manner. The biphasic regulatory effect
of ATP on PK activity was lost in PKCT, suggesting involvement
of this domain in the regulation of enzyme activity by ATP
(Figure 7B). The presence of succinyl-CoA was not required for
ATP inhibition of MRSA PK, whereas it was shown in E. coli
that ATP inhibition was dependent on succinyl-CoA (56). In-
organic phosphate (Pi) (K2HPO4-KH2PO4, pH 7.4) was a very
effective inhibitor of both PK proteins, causing 70% inhibition at
0.2 mM concentration (data not shown). Wild-type PK was
completely inactivated at 2.5 mM Pi. Thus PKCT exhibited an
activation profile comparable to that of wild-type enzyme,
indicating that the MRSA PK extra C-terminal domain is not
essential for the allosteric nature of the enzyme. However, it
might influence the magnitude of allosteric activation.
tude of activation might be explained by different affinities (S0.5
)
for substrate between the two PK proteins, so that the PEP
concentration used in the assay (5 mM) was saturating for wild
type while is subsaturating for PKCT. Furthermore, the results
indicate that activation of truncated PK by both AMP and R5P
was associated with increased cooperativity compared to that of
wild-type enzyme (Table 2). These data demonstrate a possible
regulatory role for the CT domain on allosteric activation of
MRSA PK. Both wild-type and truncated PK enzymes were not
activated by FBP, a known activator of PK enzymes (i.e., E. coli
PK1 and human M2). In fact, FBP had an inhibitory effect
(50-70%) on both proteins at 10 mM (Figure 7A,B). E. coli PK1
activation by FBP and E. coli PK2 activation by AMP and R5P
were also determined as controls (Figure 7C,D). These results
suggest that PKCT exhibited a comparable activation profile to
that of wild-type enzyme, indicating that the MRSA PK extra
C-terminal domain is not essential for the allosteric nature of the
enzyme but might regulate the magnitude of allosteric activation.
The effects of four potential inhibitors (oxalate, citrate, ATP,
and Pi) on PK activity were also evaluated. Oxalate (an analogue
In exponentially growing bacteria, ATP is present at a con-
centration of about 1.5-4 mM (42), whereas AMP and FBP
concentrations are 0.28 and 15 mM (43), respectively, and the
concentration of combined pools of pentose 5-phosphates are
1.3 mM. Results of the present study suggest that FBP and ATP
inhibition combined with AMP and R5P activation may play a
central role in regulating PK activity in MRSA.
DISCUSSION
The present study, for the first time, suggests that phospho-
fructokinase (pfk) and pyruvate kinase (pyk) constitute an
operon in S. aureus, as a survey of available genome data for a

7744 Biochemistry, Vol. 49, No. 35, 2010
Zoraghi et al.
number of S. aurues strains revealed a tandem pfk-pyk arrange-
ment. Independent expression of either PK or PFK antisense
RNAs was sufficient to induce a lethal phenotype in S. aureus
RN4420 strain (data not shown). In contrast, only PK was
identified as an essential gene through independent chromo-
somal gene knockout experiments using targetron technology
(Figure 3). These results establish the presence of a bicistronic
operon for pyk and pfk in S. aureus. A pfk-pyk operon was
previously found in some Gram-positive bacteria such as S. bovis,
L. bulgaricus, L. lactis, M. genitalium, B. stearothermophilus,
B. psychrophilus, and B. licheniformis (28, 31-35, 44). It appears
that the pfk-pyk operon may be is a Gram-positive specific
feature since no such operon association was reported in Gram-
negative bacteria. However, the biological significance of this
operon is not fully understood. Recent data suggest its involve-
ment in transcriptional regulation of carbon catabolism in Gram-
positive organisms (45).
We also provide direct evidence that S. aureus PK carries out a
function that is absolutely essential for growth and viability of
this key Gram-positive pathogen. This is based upon the finding
that inactivation of chromosomal pyk by insertion of the group II
intron at various positions of sense and antisense strands of pyk
created a lethal phenotype. PK has been previously identified as a
critical staphylococcal gene using conditional genome wide
phenotypes generated by antisense RNA (37). However, it was
not clear whether expression of PK antisense RNA resulted in
lethal isogenic or growth-defective strains. Moreover, it was
difficult from these studies to ensure gene-specific effects using
antisense technology for genes configured in polycistronic operons
where there may be polar effects. PK has been recently identified
as an absolutely essential gene for survival of other bacteria such
as H. influenzae, Streptococcus pneumoniae, and Mycobacterium
tuberculosis using allelic replacement mutagenesis (46-49). The
proven essential nature of PK in these bacteria is a very important
feature of this enzyme, since products of genes that are essential
for viability of the microorganism in vitro can be considered
as potential drug targets in the effort to develop new classes of
antimicrobial agents (50).
Our results clearly demonstrate that PK enzymatic activity is
increased during growth of S. aureus, indicating the importance
of PK for bacterial growth. Increased PK activity correlated with
increased protein expression in the early phase of growth.
However, enzyme activity dropped precipitously in the post-
exponential phase while PK protein expression was maintained.
Hence, it appears that PK activity is mainly modulated by tight
allosteric regulation, as changes in the metabolic status of the
bacteria in different phases of life cycle, particularly in the
stationary phase, result in the attenuation of PK activity regard-
less of its abundance. PK activity has previously been shown to be
important for growth of both Gram-negative (e.g., E. coli) and
Gram-positive (e.g., B. subtilis) bacteria as PK mutant strains
have substantial decreased growth rate (39, 51).
In the present study, we report for the first time a detailed
characterization of the biophysical and biochemical properties of
an active recombinant S. aureus (i.e., MRSA) PK protein.
Almost all known PKs characterized to date exist as tetramers
of four identical subunits, each of which is comprised of four
domains: N-terminal, A, B, and C domains. Our results indicate
that the active recombinant PK of S. aureus also exists as a
homotetramer. Characterization of tetramerization interactions
have been reported for a number of PKs from seven species (e.g.,
L. mexicana, E. coli, B. stearothermophilus, S. cerevisiae, human,
cat, and rabbit) (52). Tetramerization occurs through interac-
tions between A and C domains of adjacent subunits so that
bordering A domains constitute the A-A or “large” interface
and adjacent C domains form the C-C or “small” interface
between monomers. The amino acid sequences of A and B
domains, containing the predicted substrate binding sites and
cation binding sites, are well conserved in both prokaryotes
and eukaryotes. However, the C-domain is variable in some
organisms.
Our results confirmed that the predicted substrate (ADP and
PEP) binding sites and cation binding sites located in A and B
domains of bacterial PKs (e.g, MRSA PK) are well conserved
when compared with those of human isozymes (Figure 1). In
contrast, the predicted effector binding sites located in the C
domain, which are involved in allosteric regulation of PK
enzymes, are not conserved between bacteria and human iso-
forms. Some unique sequences, including a deletion, were
identified in the predicted effector binding sites of the MRSA
PK C domain (Figure 1). This is of particular interest for the
design of selective inhibitors that act specifically against bacterial
PK since selective targeting of human M2 PK containing a
unique region for allosteric regulation recently provided an
opportunity to target cell metabolism for cancer therapy despite
the high amino acid homology (95%) and identical catalytic site
of the M1 splice variant (24). This provides proof of principle
that PKs may be selectively targeted despite high degrees of
homology.
Our findings showed that both AMP and R5P (Figure 7) were
heterotropic activators of PK and R5P was slightly more potent
than AMP. We also found that MRSA PK exhibited homotropic
positive cooperativity for PEP but not for ADP (data not shown).
These findings suggest that the structure of the allosteric effector-
binding site of MRSA PK differs from those of other PKs
activated by FBP. A comparison of the predicted binding sites of
the 6-phosphate moiety of FBP, located in the C-domain of
MRSA PK, with the corresponding regions of other PKs is
shown in Figure 1. In PKs activated by FBP, a conserved lysine
(i.e., K; residue 433 in human M2) is thought to be involved in
binding the 6-phosphate moiety of FBP (11). However, similar to
nonallosteric enzymes (e.g., human M1 isozyme), MRSA PK has
a glutamate at this position (i.e., E; residue 380) (Figure 1). This
predicts that FBP should not be an allosteric activator of MRSA
PK, and this is what we found (Figure 7A, Table 2). Hence, the
observed properties of MRSA PK are consistent with the amino
acid sequence data. The fact that MRSA PK was activated by
AMP and R5P and not activated by FBP would appear to align
MRSA PK with the type II group of bacterial PKs (e.g., E. coli
PK2). However, this classification is contradicted by the higher
identities at the amino acid sequence level with the type I group of
bacterial PKs (e.g., 48% E. coli PK1).
Despite similarities in allosteric behavior to type II PKs,
MRSA PK showed several interesting distinguishing properties.
One feature of MRSA PK is that the enzymatic activity was
inhibited at physiologic concentrations of FBP (Figure 6A). FBP
is well-known to be an allosteric activator of most PKs including
E. coli PK1 and several human PKs. To our knowledge a negative
effect of FBP on any bacterial PK has been reported. Further
investigations are necessary to clarify the physiological signifi-
cance of the inhibitory effect of FBP on MRSA PK.
MRSA PK is the only bacterial PK characterized to date to
show a dual response to ATP, as its activity was stimulated by
low concentrations of ATP (e.g., 1 mM), but was completely

Article
Biochemistry, Vol. 49, No. 35, 2010 7745
inhibited at higher concentrations of ATP (>2.5 mM) (Figure 7A,
Table 2). ATP has previously been shown to regulate PKs via
a product feedback inhibition mechanism. On the other hand,
ATP is known to be capable of chelating magnesium ions, which
are necessary for PK enzymatic activity. Hence, the dual effects
of ATP on MRSA PK are complex and difficult to analyze
unambiguously. Although succinyl-CoA was shown to be neces-
sary for ATP inhibition in E. coli (21), it is noteworthy that
inhibition of the MRSA enzyme by ATP did not require the
presence of succinyl-CoA (Figure 7, Table 2).
domain and are regulated by AMP and R5P but not FBP and
glucose 6-phosphate (16, 28). However, CT truncation in
B. stearothermophilus does not significantly alter the efficiency of
the enzyme (kcat/S0.5) (29). In striking contrast, CT domain-deleted
mutant MRSA PK exhibited dramatically lowered enzyme
efficiency with kcat/S0.5 values less than 4.2% and 6.3% of those
for PKWT toward PEP and ADP, respectively, in the ligand-free
state. However, enzyme efficiency of truncated MRSA PK was
increased 3-fold in AMP-bound states, toward PEP. This in-
crease in mutant enzymatic activity is most likely related to
enhanced stability and rigidity of the protein upon effector
binding. Such an effect has been previously reported for another
CT-containing bacterial PK protein, B. stearothermophilus PK,
as addition of the allosteric activator R5P resulted in a highly
thermal stable PK conformation (54). These data are consistent
with the potential requirement of CT domain interactions for
optimal stability and rigidity of MRSA PK structure leading to
efficient catalytic activity. This may involve enabling stronger
interactions between adjacent domains of the tetramer, which are
thought to be essential for subunit interactions. The recent crystal
structure of B. stearothermophilus provided insight into the
subunit interface between the CT domain and A domain of
neighboring subunits. This appeared to involve the amide nitro-
gen of Leu538 in the CT domain of one subunit and the carboxyl
oxygen of Glu209 in the A domain of the neighboring subunit to
Based on our findings, a model of MRSA PK regulation might
look something like this: during exponential growth of bacteria,
ATP is rapidly depleted, and due to the shift of the Vmax in the
presence of the activator AMP (280 μM) and R5P (43), PK
becomes highly active at the prevalent low PEP concentrations.
By the time cells reach the stationary growth phase, the lack of
readily available sugars has induced a reduction in the pentose
phosphate pathway activity leading to depletion in the level of
intracellular R5P. With the decreased energy requirement asso-
ciated with the slowdown of cell metabolism, the level of ATP
increases, which induces inhibition of the PK activity. This in
turn induces an increase in PEP concentration, thus providing for
a continued source of energy in the context of residual PK activity
leading to the production of ATP. Should a carbohydrate
substrate become once more available to the cell, owing to the
high potential of R5P as an activator, PK is able to resume a high
level of activity, thereby providing ATP for glycolysis and the
restoration of normal cell functions. Thus, PK plays a key role in
the modulation of glycolysis and cell metabolism in bacteria.
A remarkable characteristic of staphylococcal PKs (e.g.,
MRSA PK) and a few other bacteria (e.g., Bacillus sp.,
Listeria sp.) is a conserved PEP-utilizer superfamily domain,
which exists as an extra C-terminal (CT) sequence. This finding
suggested that some interaction could take place between PEP
and the CT domain. This domain in B. stearothermophilus has
been suggested to be derived from a PEP synthase late in the
evolutionary process (53). However, the biological significance of
this CT domain has not been fully elucidated. Since PKs from
other sources lack the sequence, it is unlikely that it is essential for
enzymatic activity or allosteric regulation. In this study, we
examined in detail the influence of the CT domain on enzyme
structure and function by characterizing a MRSA PK mutant
(PKCT) lacking the sequence. The expression level and the
oligomeric state of the PKCT were identical to those of the
wild-type enzyme. The truncated enzyme exhibited PK activity
similar to that of the wild-type protein, indicating that the CT
domain is not essential for the activity or the allosteric nature of
the enzyme. However, the S0.5 values for PEP in the absence and
presence of AMP and the concentration of AMP and R5P
required for half-maximal activation of PKCT were increased
by 1.6-, 3.6-, 4.6-, and 24-fold (Tables 1 and 2), respectively,
compared to the wild-type enzyme. Moreover, the stimulatory
effect of low concentrations of ATP on enzyme activity was lost
in PKCT. These findings strongly suggest that potential interac-
tions between the MRSA PK CT domain and the A and C
domains may contribute to structural stability of the holomeric
enzyme or to the specificity of the allosteric effectors of the
enzyme, or both.
˚
form a hydrogen bond (distance, 2.82 A) in B. stearothermophilus
PK (6). Leu538 and Glu209 in B. stearothermophilus PK are
conserved as Leu536 and Glu207 in MRSA PK. The impact of
the CT domain in stability and rigidity of the MRSA PK tetramer
is of particular interest since the results of several published site-
directed mutagenesis studies on PKs showed that single muta-
tions located along the effector binding sites distant from the
active sites can have unpredictably profound effects on the rates
of enzyme catalysis. It is quite possible and highly likely that these
effects may be due to altered dynamics of domains leading to
possibly altered stability and rigidity of the tetramer (1, 55-58).
Nevertheless, the evolutionary advantage of the CT domain for
S. aureus PK, if any, is not clear at this time. The PEP binding
motif may participate in some as yet unknown enzymatic reac-
tion. The possibility that the conserved histidine residues in the
PEP-utilizer superfamily binding motif of MRSA PK are phos-
phorylated by PEP is, however, highly unlikely since it is replaced
by proline at position 539 (Figure 1). The PEP binding motif may
also participate in some as yet unknown enzymatic reaction.
The findings reported above provide novel insights into the
functional features and mechanisms of regulation of S. aureus PK
and its potential as an MRSA drug target. The essential nature of
MRSA PK, the correlation of its peak enzymatic activity with
exponential growth phase, its unique kinetic properties, and
distinct allosteric regulation when compared with human isoforms
suggest that it may be an attractive, novel drug target. This notion
is supported further by the high level of sequence and structural
divergence defined by biocomputational analysis between bacterial
and mammalian PKs (Cherkasov et al., unpublished results).
Currently, the availability of copious amounts of recombinant
MRSA PK will facilitate both X-ray crystallographic studies
aimed at the design of small molecules that selectively inhibit
MRSA PK and screening of candidates in enzymatic assays.
Sequence identity (68%), the presence of an extra C-terminal
domain, kinetic properties, and mode of allosteric regulation of
MRSA PK most closely resemble PK from B. stearothermophilus.
Both MRSA and B. stearothermophilus PKs contain the CT
ACKNOWLEDGMENT
Special thanks to Mrs. Marija Vuckovic from Dr. Natalie
Strynadka’s laboratory for help with size exclusion chromatography.

7746 Biochemistry, Vol. 49, No. 35, 2010
Zoraghi et al.
REFERENCES
Identification of small molecule inhibitors of pyruvate kinase M2.
Biochem. Pharmacol. 79, 1118–1124.
1. Valentini, G., Chiarelli, L., Fortin, R., Speranza, M. L., Galizzi, A.,
and Mattevi, A. (2000) The allosteric regulation of pyruvate kinase.
J. Biol. Chem. 275, 18145–18152.
2. Larsen, T. M., Laughlin, L. T., Holden, H. M., Rayment, I., and Reed,
G. H. (1994) Structure of rabbit muscle pyruvate kinase complexed
with Mn2þ, Kþ, and pyruvate. Biochemistry 33, 6301–6309.
3. Jurica, M. S., Mesecar, A., Heath, P. J., Shi, W., Nowak, T., and
Stoddard, B. L. (1998) The allosteric regulation of pyruvate kinase by
fructose-1,6-bisphosphate. Structure 6, 195–210.
4. Speranza, M. L., Valentini, G., Iadarola, P., Stoppini, M., Malcovati,
M., and Ferri, G. (1989) Primary structure of three peptides at the
catalytic and allosteric sites of the fructose-1,6-bisphosphate-acti-
vated pyruvate kinase from Escherichia coli. Biol. Chem. Hoppe-
Seyler 370, 211–216.
5. Rigden, D. J., Phillips, S. E., Michels, P. A., and Fothergill-Gilmore,
L. A. (1999) The structure of pyruvate kinase from Leishmania
mexicana reveals details of the allosteric transition and unusual
effector specificity. J. Mol. Biol. 291, 615–635.
25. Nowicki, M. W., Tulloch, L. B., Worralll, L., McNae, I. W., Hannaert,
V., Michels, P. A., Fothergill-Gilmore, L. A., Walkinshaw, M. D.,
and Turner, N. J. (2008) Design, synthesis and trypanocidal activity of
lead compounds based on inhibitors of parasite glycolysis. Bioorg.
Med. Chem. 16, 5050–5061.
26. Chan, M., Tan, D. S., and Sim, T. S. (2007) Plasmodium falciparum
pyruvate kinase as a novel target for antimalarial drug-screening.
Travel Med. Infect. Dis. 5, 125–131.
27. Garcia-Lara, J., Masalha, M., and Foster, S. J. (2005) Staphylococcus
aureus: the search for novel targets. Drug Discovery Today 10,
643–651.
28. Sakai, H., and Ohta, T. (1993) Molecular cloning and nucleotide
sequence of the gene for pyruvate kinase of Bacillus stearothermophi-
lus and the production of the enzyme in Escherichia coli. Evidence that
the genes for phosphofructokinase and pyruvate kinase constitute an
operon. Eur. J. Biochem. 211, 851–859.
29. Sakai, H. (2004) Possible structure and function of the extra C-term-
inal sequence of pyruvate kinase from Bacillus stearothermophilus.
J. Biochem. 136, 471–476.
6. Suzuki, K., Ito, S., Shimizu-Ibuka, A., and Sakai, H. (2008) Crystal
structure of pyruvate kinase from Geobacillus stearothermophilus.
J. Biochem. 144, 305–312.
7. Stuart, D. I., Levine, M., Muirhead, H., and Stammers, D. K. (1979)
Crystal structure of cat muscle pyruvate kinase at a resolution of 2.6
˚
A. J. Mol. Biol. 134, 109–142.
30. Yao, J., Zhong, J., Fang, Y., Geisinger, E., Novick, R. P., and
Lambowitz, A. M. (2006) Use of targetrons to disrupt essential and
nonessential genes in Staphylococcus aureus reveals temperature
sensitivity of Ll.LtrB group II intron splicing. RNA 12, 1271–1281.
31. Branny, P., De La Torre, F., and Garel, J. R. (1993) Cloning,
sequencing, and expression in Escherichia coli of the gene coding for
phosphofructokinase in Lactobacillus bulgaricus. J. Bacteriol. 175,
5344–5349.
8. Valentini, G., Chiarelli, L. R., Fortin, R., Dolzan, M., Galizzi, A.,
Abraham, D. J., Wang, C., Bianchi, P., Zanella, A., and Mattevi, A.
(2002) Structure and function of human erythrocyte pyruvate kinase.
Molecular basis of nonspherocytic hemolytic anemia. J. Biol. Chem.
277, 23807–23814.
9. Munoz, M. E., and Ponce, E. (2003) Pyruvate kinase: current status of
regulatory and functional properties. Comp. Biochem. Physiol. B: Biochem.
Mol. Biol. 135, 197–218.
10. Noguchi, T., Inoue, H., and Tanaka, T. (1986) The M1- and M2-type
isozymes of rat pyruvate kinase are produced from the same gene by
alternative RNA splicing. J. Biol. Chem. 261, 13807–13812.
11. Dombrauckas, J. D., Santarsiero, B. D., and Mesecar, A. D. (2005)
Structural basis for tumor pyruvate kinase M2 allosteric regulation
and catalysis. Biochemistry 44, 9417–9429.
12. van Schaftingen, E., Opperdoes, F. R., and Hers, H. G. (1985)
Stimulation of Trypanosoma brucei pyruvate kinase by fructose 2,6-
bisphosphate. Eur. J. Biochem. 153, 403–406.
13. Iliffe-Lee, E. R., and McClarty, G. (2002) Pyruvate kinase from
Chlamydia trachomatis is activated by fructose-2,6-bisphosphate.
Mol. Microbiol. 44, 819–828.
14. Morgan, H. P., McNae, I. W., Nowicki, M. W., Hannaert, V.,
Michels, P. A., Fothergill-Gilmore, L. A., and Walkinshaw, M. D.
(2010) The allosteric mechanism of pryuvate kinase from Leishmania
mexicana: a rock and lock model, J. Biol. Chem. (in press).
15. Malcovati, M., and Valentini, G. (1982) AMP- and fructose 1,6-
bisphosphate-activated pyruvate kinases from Escherichia coli. Meth-
ods Enzymol. 90 (Part E), 170–179.
16. Sakai, H., Suzuki, K., and Imahori, K. (1986) Purification and
properties of pyruvate kinase from Bacillus stearothermophilus. J. Biochem.
99, 1157–1167.
17. Chuang, D. T., and Utter, M. F. (1979) Structural and regulatory
properties of pyruvate kinase from Pseudomonas citronellolis. J. Biol.
Chem. 254, 8434–8441.
32. Branny, P., De La Torre, F., and Garel, J. R. (1996) The genes for
phosphofructokinase and pyruvate kinase of Lactobacillus delbrueckii
subsp. bulgaricus constitute an operon. J. Bacteriol. 178, 4727–4730.
33. Llanos, R. M., Harris, C. J., Hillier, A. J., and Davidson, B. E. (1993)
Identification of a novel operon in Lactococcus lactis encoding three
enzymes for lactic acid synthesis: phosphofructokinase, pyruvate
kinase, and lactate dehydrogenase. J. Bacteriol. 175, 2541–2551.
34. Fraser, C. M., Gocayne, J. D., White, O., Adams, M. D., Clayton,
R. A., Fleischmann, R. D., Bult, C. J., Kerlavage, A. R., Sutton, G.,
Kelley, J. M., Fritchman, R. D., Weidman, J. F., Small, K. V.,
Sandusky, M., Fuhrmann, J., Nguyen, D., Utterback, T. R., Saudek,
D. M., Phillips, C. A., Merrick, J. M., Tomb, J. F., Dougherty, B. A.,
Bott, K. F., Hu, P. C., Lucier, T. S., Peterson, S. N., Smith, H. O.,
Hutchison, C. A., III, and Venter, J. C. (1995) The minimal gene
complement of Mycoplasma genitalium. Science 270, 397–403.
35. Tanaka, K., Sakai, H., Ohta, T., and Matsuzawa, H. (1995) Molec-
ular cloning of the genes for pyruvate kinase of two bacilli, Bacillus
psychrophilus and Bacillus licheniformis, and comparison of the
properties of the enzymes produced in Escherichia coli. Biosci.,
Biotechnol., Biochem. 59, 1536–1542.
36. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J.
(1990) Basic local alignment search tool. J. Mol. Biol. 215, 403–410.
37. Forsyth, R. A., Haselbeck, R. J., Ohlsen, K. L., Yamamoto, R. T.,
Xu, H., Trawick, J. D., Wall, D., Wang, L., Brown-Driver, V.,
Froelich, J. M., C, K. G., King, P., McCarthy, M., Malone, C.,
Misiner, B., Robbins, D., Tan, Z., Zhu Zy, Z. Y., Carr, G., Mosca,
D. A., Zamudio, C., Foulkes, J. G., and Zyskind, J. W. (2002) A
genome-wide strategy for the identification of essential genes in
Staphylococcus aureus. Mol. Microbiol. 43, 1387–1400.
38. Rodriguez, S. A., Davis, G., and Klose, K. E. (2009) Targeted gene
disruption in Francisella tularensis by group II introns. Methods 49,
270–274.
39. Somerville, G. A., Chaussee, M. S., Morgan, C. I., Fitzgerald, J. R.,
Dorward, D. W., Reitzer, L. J., and Musser, J. M. (2002) Staphylo-
coccus aureus aconitase inactivation unexpectedly inhibits post-ex-
ponential-phase growth and enhances stationary-phase survival.
Infect. Immun. 70, 6373–6382.
18. Crow, V. L., and Pritchard, G. G. (1982) Pyruvate kinase from
Streptococcus lactis. Methods Enzymol. 90 (Part E), 165–170.
19. Le Bras, G., and Garel, J. R. (1993) Pyruvate kinase from Lactoba-
cillus bulgaricus: possible regulation by competition between strong
and weak effectors. Biochimie 75, 797–802.
20. Kapoor, R., and Venkitasubramanian, T. A. (1983) Purification and
properties of pyruvate kinase from Mycobacterium smegmatis. Arch.
Biochem. Biophys. 225, 320–330.
40. Fry, B., Zhu, T., Domach, M. M., Koepsel, R. R., Phalakornkule, C.,
and Ataai, M. M. (2000) Characterization of growth and acid
formation in a Bacillus subtilis pyruvate kinase mutant. Appl. Environ.
Microbiol. 66, 4045–4049.
21. Waygood, E. B., and Sanwal, B. D. (1974) The control of pyruvate
kinases of Escherichia coli. I. Physicochemical and regulatory proper-
ties of the enzyme activated by fructose 1,6-diphosphate. J. Biol.
Chem. 249, 265–274.
41. Abbe, K., and Yamada, T. (1982) Purification and properties of
pyruvate kinase from Streptococcus mutans. J. Bacteriol. 149, 299–305.
42. Schaub, J., Schiesling, C., Reuss, M., and Dauner, M. (2006) Inte-
grated sampling procedure for metabolome analysis. Biotechnol.
Prog. 22, 1434–1442.
43. Bennett, B. D., Kimball, E. H., Gao, M., Osterhout, R., Van Dien,
S. J., and Rabinowitz, J. D. (2009) Absolute metabolite concentra-
tions and implied enzyme active site occupancy in Escherichia coli.
Nat. Chem. Biol. 5, 593–599.
22. Opperdoes, F. R., and Michels, P. A. (2001) Enzymes of carbohydrate
metabolism as potential drug targets. Int. J. Parasitol. 31, 482–490.
23. Christofk, H. R., Vander Heiden, M. G., Harris, M. H., Ramanathan,
A., Gerszten, R. E., Wei, R., Fleming, M. D., Schreiber, S. L., and
Cantley, L. C. (2008) The M2 splice isoform of pyruvate kinase is
important for cancer metabolism and tumour growth. Nature 452,
230–233.
24. Vander Heiden, M. G., Christofk, H. R., Schuman, E., Subtelny,
A. O., Sharfi, H., Harlow, E. E., Xian, J., and Cantley, L. C. (2010)

Article
Biochemistry, Vol. 49, No. 35, 2010 7747
44. Asanuma, N., Kanada, K., and Hino, T. (2008) Molecular properties
and transcriptional control of the phosphofructokinase and pyruvate
kinase genes in a ruminal bacterium, Streptococcus bovis. Anaerobe
14, 237–241.
45. Kowalczyk, M., and Bardowski, J. (2007) Regulation of sugar
catabolism in Lactococcus lactis. Crit. Rev. Microbiol. 33, 1–13.
46. Zhang, R., Ou, H. Y., and Zhang, C. T. (2004) DEG: a database of
essential genes. Nucleic Acids Res. 32, D271–272.
induces a major conformational change in Leishmania mexicana
pyruvate kinase in the crystalline state. J. Mol. Biol. 383, 615–626.
53. Pocalyko, D. J., Carroll, L. J., Martin, B. M., Babbitt, P. C., and
Dunaway-Mariano, D. (1990) Analysis of sequence homologies in
plant and bacterial pyruvate phosphate dikinase, enzyme I of the
bacterial phosphoenolpyruvate:sugar phosphotransferase system and
other PEP-utilizing enzymes. Identification of potential catalytic and
regulatory motifs. Biochemistry 29, 10757–10765.
47. Akerley, B. J., Rubin, E. J., Novick, V. L., Amaya, K., Judson, N.,
and Mekalanos, J. J. (2002) A genome-scale analysis for identification
of genes required for growth or survival of Haemophilus influenzae.
Proc. Natl. Acad. Sci. U.S.A. 99, 966–971.
48. Song, J. H., Ko, K. S., Lee, J. Y., Baek, J. Y., Oh, W. S., Yoon, H. S.,
Jeong, J. Y., and Chun, J. (2005) Identification of essential genes in
Streptococcus pneumoniae by allelic replacement mutagenesis. Mol.
Cells 19, 365–374.
49. Sassetti, C. M., Boyd, D. H., and Rubin, E. J. (2003) Genes required
for mycobacterial growth defined by high density mutagenesis. Mol.
Microbiol. 48, 77–84.
50. Thomson, C. J., Power, E., Ruebsamen-Waigmann, H., and La-
bischinski, H. (2004) Antibacterial research and development in the
21(st) century—an industry perspective of the challenges. Curr. Opin.
Microbiol. 7, 445–450.
54. Lovell, S. C., Mullick, A. H., and Muirhead, H. (1998) Cooperativity
in Bacillus stearothermophilus pyruvate kinase. J. Mol. Biol. 276,
839–851.
55. Hannaert, V., Yernaux, C., Rigden, D. J., Fothergill-Gilmore, L. A.,
Opperdoes, F. R., and Michels, P. A. (2002) The putative effector-
binding site of Leishmania mexicana pyruvate kinase studied by site-
directed mutagenesis. FEBS Lett. 514, 255–259.
56. Ikeda, Y., Tanaka, T., and Noguchi, T. (1997) Conversion of non-
allosteric pyruvate kinase isozyme into an allosteric enzyme by a single
amino acid substitution. J. Biol. Chem. 272, 20495–20501.
57. Collins, R. A., Kelly, S. M., Price, N. C., Fothergill-Gilmore, L. A.,
and Muirhead, H. (1996) Ligand-induced conformational changes in
wild-type and mutant yeast pyruvate kinase. Protein Eng. 9, 1203–
1210.
58. Mattevi, A., Valentini, G., Rizzi, M., Speranza, M. L., Bolognesi, M.,
and Coda, A. (1995) Crystal structure of Escherichia coli pyruvate kinase
type I: molecular basis of the allosteric transition. Structure 3, 729–741.
59. Dereeper, A., Guignon, V., Blanc, G., Audic, S., Buffet, S., Chevenet,
F., Dufayard, J. F., Guindon, S., Lefort, V., Lescot, M., Claverie,
J. M., and Gascuel, O. (2008) Phylogeny.fr: robust phylogenetic
analysis for the non-specialist. Nucleic Acids Res. 36, W465–469.
51. Cunningham, D. S., Liu, Z., Domagalski, N., Koepsel, R. R., Ataai,
M. M., and Domach, M. M. (2009) Pyruvate kinase-deficient Escher-
ichia coli exhibits increased plasmid copy number and cyclic AMP
levels. J. Bacteriol. 191, 3041–3049.
52. Tulloch, L. B., Morgan, H. P., Hannaert, V., Michels, P. A., Fothergill-
Gilmore, L. A., and Walkinshaw, M. D. (2008) Sulphate removal