Alternative substrates selective for S-adenosylmethionine synthetases from pathogenic bacteria

Article abstract of DOI:10.1016/j.abb.2013.05.008

S-adenosyl-L-methionine (AdoMet) synthetase catalyzes the production of AdoMet, the major biological methyl donor and source of methylene, amino, ribosyl, and aminopropyl groups in the metabolism of all known organism. In addition to these essential functions, AdoMet can also serve as the precursor for two different families of quorum sensing molecules that trigger virulence in Gram-negative human pathogenic bacteria. The enzyme responsible for AdoMet biosynthesis has been cloned, expressed and purified from several of these infectious bacteria. AdoMet synthetase (MAT) from Neisseria meningitidis shows similar kinetic parameters to the previously characterized Escherichia coli enzyme, while the Pseudomonas aeruginosa enzyme has a decreased catalytic efficiency for its MgATP substrate. In contrast, the more distantly related MAT from Campylobacter jejuni has an altered quaternary structure and possesses a higher catalytic turnover than the more closely related family members. Methionine analogs have been examined to delineate the substrate specificity of these enzyme forms, and several alternative substrates have been identified with the potential to block quorum sensing while still serving as precursors for essential methyl donation and radical generation reactions.

Full text of DOI:10.1016/j.abb.2013.05.008

Archives of Biochemistry and Biophysics 536 (2013) 64–71
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journal homepage: www.elsevier.com/locate/yabbi
Alternative substrates selective for S-adenosylmethionine synthetases
from pathogenic bacteria
1
⇑
Stephen P. Zano, Pravin Bhansali, Amarjit Luniwal , Ronald E. Viola
Department of Chemistry, The University of Toledo, Toledo, OH 43606, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
S-adenosyl-L-methionine (AdoMet) synthetase catalyzes the production of AdoMet, the major biological
Received 19 February 2013
and in revised form 7 May 2013
Available online 24 May 2013
methyl donor and source of methylene, amino, ribosyl, and aminopropyl groups in the metabolism of all
known organism. In addition to these essential functions, AdoMet can also serve as the precursor for two
different families of quorum sensing molecules that trigger virulence in Gram-negative human patho-
genic bacteria. The enzyme responsible for AdoMet biosynthesis has been cloned, expressed and purified
from several of these infectious bacteria. AdoMet synthetase (MAT) from Neisseria meningitidis shows
similar kinetic parameters to the previously characterized Escherichia coli enzyme, while the Pseudomonas
aeruginosa enzyme has a decreased catalytic efficiency for its MgATP substrate. In contrast, the more dis-
tantly related MAT from Campylobacter jejuni has an altered quaternary structure and possesses a higher
catalytic turnover than the more closely related family members. Methionine analogs have been exam-
ined to delineate the substrate specificity of these enzyme forms, and several alternative substrates have
been identified with the potential to block quorum sensing while still serving as precursors for essential
methyl donation and radical generation reactions.
Keywords:
Alternative substrates
Enzyme kinetics
AdoMet synthetase
Pathogenic bacteria
Quorum sensing
Ó 2013 Elsevier Inc. All rights reserved.
Introduction
synthase [5]. The ribosyl group of AdoMet is a precursor in the next
to last step of the biosynthesis pathway to a modified tRNA nucle-
The only known route for AdoMet biosynthesis is catalyzed by
ATP: -methionine S-adenosyltransferase ( -methionine adenosyl-
synthetase or MAT, EC 2.5.1.6). AdoMet synthesis occurs via an
unusual two-step reaction. First, -methionine reacts with ATP to
yield the sulfonium compound, S-adenosyl- -methionine (Ado-
Met), and tripolyphosphate (PPPi) through an SN2 mechanism [1].
In the second step, the PPPi is asymmetrically hydrolyzed to inor-
ganic pyrophosphate (PPi) and orthophosphate (Pi) as the final
products [2]. Sequence homology of eukarya and bacterial MATs
reveal a well-conserved 380–400 residue protein family. In partic-
ular, the residues present in the active site of each of the reported
AdoMet synthetases are fully conserved [3].
oside [6]. AdoMet is also a donor of aminopropyl groups to produce
diamines during polyamine biosynthesis. The 50-deoxyadenosyl
moiety of AdoMet is utilized in a range of free radical reactions that
include biosynthesis of deoxyribonucleotides, and glycyl radical-
generating reactions [7]. This diversity of group transfer reactions
places AdoMet at a key intersection of amino acid, nucleic acid
and lipid metabolism. Consistent with these critical functions,
the methionine metabolic cycle that is involved in the synthesis
of AdoMet [8] is tightly regulated at both the genetic and protein
levels [9,10].
In addition to these essential roles in mammalian metabolism,
AdoMet is also a precursor for the production of two classes of quo-
rum sensing (QS)2 molecules in certain bacteria [11]. These com-
pounds regulate the expression of a large number of bacterial
genes, including those that trigger virulence in human pathogenic
organisms [12]. Many of these quorum-responsive genes produce
virulence factors, including secreted toxins, proteases and hemoly-
sins that cause disease pathology, as well as components required
for assembly of the polysaccharide matrix of biofilms that protect
microbes against phagocytes and antibiotics [13]. Because of the
central role of AdoMet there has been considerable interest in
L
L
L
L
The product of the enzyme-catalyzed reaction, S-adenosyl-L-
methinonine, also known as SAM or AdoMet, is an important bio-
logical sulfonium compound that plays a central role in many
essential biochemical processes in all known organism. AdoMet
functions as a donor in the methylation reactions of DNA, RNA
and proteins as well as for small molecules such as phospholipids
and various neurotransmitters [4]. The amino group of AdoMet is
also donated in the biotin biosynthetic pathway catalyzed by DAPA
⇑
Corresponding author. Fax: +1 419 530 1583.
E-mail address: ron.viola@utoledo.edu (R.E. Viola).
Current address: NAMSA, 6750 Wales Road, Northwood, OH 43619.
2
Abbreviations used: QS, quorum sensing; IPTG, isopropyl b-D-thiogalactopyrano-
1
side; DTT, dithiothreitol; DLS, dynamic light scattering; MAT, AdoMet synthetase.
0003-9861/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.abb.2013.05.008

S.P. Zano et al. / Archives of Biochemistry and Biophysics 536 (2013) 64–71
65
examining the trafficking of this metabolite for the development of
anticancer [14] and antiviral drugs [15]. However, it has been diffi-
cult to achieve the selectivity required to target pathogenic functions
while also minimizing disruption of the essential mammalian meta-
bolic functions of AdoMet.
were cloned into pET101/D-TOPO using the TOPO directional clon-
ing kit from Invitrogen. The coupling enzymes and substrates used
for the enzyme activity assay were obtained from Sigma–Aldrich.
Synthesis of methionine derivatives
To overcome this difficulty, we have focused on the synthetic
machinery of AdoMet, the S-adenosylmethionine synthetases
(MATs) from several human pathogenic organisms as targets for
antibacterial development. Pseudomonas aeruginosa causes gastro-
intestinal and urinary tract infections and is the most common
cause of infections in burn patients [16]. This pathogen has devel-
oped multidrug resistance to many common antibiotics [17] and
can grow into biofilms associated with enhanced resistance to anti-
biotic treatment [18]. Neisseria meningitidis, a meningococcus, is a
strictly human pathogen that can cause a range of serious diseases
once it penetrates the mucosal membrane and enters the blood-
stream. Infection by this organism can cause meningitis, a severe
sepsis that is often fatal and, more rarely, can lead to other diseases
such as septic arthritis, pneumonia, purulent pericarditis, conjunc-
tivitis, otitis, sinusitis and urethritis [19]. Drug-resistant meningo-
cocci were reported as early as 1960; a consequence of horizontal
gene transfer and point mutations [20], and the overuse and mis-
use of antibiotics has further exacerbated the drug-resistance
problem. Campylobacter jejuni, along with Salmonella, are the most
frequent causes of food poisoning [21]. While infections from this
organism are rarely life-threatening, they have been linked with
subsequent development of peripheral neuropathies such as
Guillain–Barre syndrome [22] that can develop two to three weeks
after the initial infection. These target bacterial organisms and
related Gram-negative pathogens use the production of QS mole-
cules called autoinducers to link expression of their virulence
properties to population density [23]. Selective interference with
these QS signaling pathways would examine an underexplored
new approach for infection control through the generation of
anti-virulence but not bactericidal compounds that could have
much lower selection pressure for the development of drug
resistance.
This work reports the production and characterization of Ado-
Met synthetases from several human pathogenic organisms, P.
aeruginosa, N. meningitidis, and C. jejuni, along with the orthologous
enzyme from Escherichia coli. To test the viability of this enzyme
target for drug development, alternative substrates with species
selectivity have been identified for the AdoMet synthetases (MATs)
from these target organisms. The products obtained from these
alternative reactions are AdoMet analogs with the potential to dis-
criminate between essential mammalian metabolic functions and
pathogenic triggering activities. Differences in the efficiency by
which these enzymes utilize these alternative substrates suggest
subtle differences in substrate recognition that could be further
exploited for the development of species-specific quorum sensing
inhibition.
Synthesis of
L
-methionine phenyl ester was started by conver-
sion of Boc- -methionine p-nitrophenyl ester to Boc-
L
L
-methionine
phenyl ester using phenol. Deprotection of the amino group was
achieved using trifluoroacetic acid/dichloromethane mixture to
obtain L-methionine phenyl ester. The structure of the phenyl ester
was confirmed by NMR (1H NMR (600 MHz, CD3OD): d = 7.15–7.17
(dd, J = 7.44, 8.52 Hz, 2H), 6.77–6.80 (m, 3H), 4.11 (t, J = 5.88 Hz,
1H), 3.33 (s, 1H), 2.65 (t, J = 7.08 Hz, 2H), 2.19–2.24 (m, 1H),
2.07–2.13 (m, 4H) ppm. 13C NMR (150 MHz, CD3OD): d = 172.5,
158.5, 130.4, 120.5, 116.3, 53.7, 31.5, 30.3, 15.0 ppm). Similarly,
L
-methionine methyl ester was synthesized by reacting L-methio-
nine with methanol in presence of catalytic concentrated sulphuric
acid under reflux (1H NMR (600 MHz, DMSO) d = 8.83 (br, 3H), 4.07
(t, J = 6 Hz, 1H), 3.73 (s, 3H), 2.65 (m, 1H), 2.53 (m, 1H), 2.09 (m,
2H), 2.04 (s, 3H). 13C NMR (150 MHz, DMSO) d = 169.6, 52.8, 50.7,
29.3, 28.3, 14.2). Synthesis of N,N-Dimethyl-
ester was achieved under reductive amination conditions. First,
methionine methyl ester was reacted with formaldehyde and
was then reduced in situ to the dimethylamine derivative using so-
dium cyanoborohydride to obtain N,N-dimethyl methionine
methyl ester (1H NMR (600 MHz, CDCl3) d = 3.72 (s, 3H), 3.33 (t,
J = 12 Hz, 1H), 2.54 (m, 2H), 2.34 (s, 6H), 2.10 (s, 3H), 2.00 (m,
1H), 1.92 (m, 1H). 13C NMR (150 MHz, CDCl3) d = 172.3, 65.8,
51.1, 41.5, 30.7, 28.6, 15.4). The latter was then hydrolyzed to
N,N-dimethyl methionine under acidic conditions.
L-methionine methyl
L-
Enzyme purification
The open reading frame (ORF) of AdoMet synthetases (metK)
were expressed in pET DEST42 or pET101/D-TOPO vector that
introduces a C-terminal V5 epitope and hexahistidine tag on the
protein. Positive expression clones were transformed into
BL21(DE3) E. coli cells for expression. Four liters of LB media con-
taining ampicillin (100 lg/mL) were inoculated and grown at
37 °C until A600 P 0.75, then gene expression was induced with
1 mM isopropyl b-D-thiogalactopyranoside (IPTG) and further
grown for 4 h at 28 °C. Cell paste was collected by centrifugation
at 15,300g for 15 min using a JA-14 rotor in a Beckman J2-HS
refrigerated centrifuge. Collected cell pellets were stored at
ꢀ80 °C until use.
Five grams of cell paste were resuspended in Buffer A, com-
posed of 50 mM Tris–HCl, pH 8.0, 300 mM KCl, 5 mM b-mercap-
toethanol, 5% glycerol and 25 mM imidazole. The resuspended
cells were lysed by ultrasonication using a 30 s pulse on and
2 min pulse off protocol for a total of 8 min. The clarified soluble
lysate were loaded onto a 20 mL Ni-IMAC column equilibrated
with 4 column volumes of Buffer A using an ÄKTA chromatography
system. After washing the column with 3 column volumes of Buffer
A, the bound enzyme was then eluted by a 300 mL linear gradient
with Buffer A containing 400 mM imidazole (25 mM to 400 mM
imidazole gradient). Fractions showing AdoMet synthetase activity
were pooled and dialyzed against Buffer B, composed of 50 mM
Tris–HCl, pH 8.0, 50 mM KCl and 1.0 mM dithiothreitol (DTT).
These protein samples were then loaded onto a 20 mL Source
30Q anion exchange column and the bound enzyme was eluted
with a 300 mL linear gradient of Buffer B containing 500 mM KCl.
Each step in the purification was monitored by SDS gel electropho-
resis on an Invitrogen XCell Surelock Mini-Cell Electrophoresis Sys-
tem using 4–16% Bis–Tris gradient gels and MES running buffer. All
Experimental procedures
Materials and bacterial strains
All reagents used were of highest purity commercially available.
Methionine derivatives and analogs were either purchased from
Chem-Impex International or were synthesized as described be-
low. DNA polymerase and restriction enzymes were purchased
from New England Biolabs while plasmid miniprep kits and gel
extraction kits were from Qiagen. The Gateway cloning technology
kit (Life Technologies) was used for cloning of the E. coli K12 and P.
aeruginosa PA01 metK genes into the pET DEST42 expression vec-
tor. The N. meningitidis WUE2594 and C. jejuni 81-176 metK genes

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S.P. Zano et al. / Archives of Biochemistry and Biophysics 536 (2013) 64–71
protein concentrations were determined by using protein A280
with a NanoDrop 2000 UV–Vis Spectrophotometer.
pyrophosphorylase. The catalytic activity of each coupling reaction
was separately optimized to ensure that the production of AdoMet
or AdoMet analogs catalyzed by the respective AdoMet synthetases
is the rate limiting step in this assay.
Protein characterization
The kinetic parameters for both substrates, ATP and
nine, were determined by varying their concentration from
10 M to 1.0 mM and measuring the production of NADPH at
L-methio-
Mass spectrometry was performed to confirm the identity of
the purified AdoMet synthetases using a Bruker ultrafleXtreme
l
340 nm. The values obtained for the E. coli enzyme are comparable
to those reported for this enzyme by using the radiolabeled assay
[1], confirming the validity of this coupled assay. Parameters for
the alternative substrates were evaluated by varying their concen-
trations to determine the KM values. Substrate data were evaluated
by adapting the kinetic equations of Cleland [26]. The inhibition
MALDI-TOF/TOF Spectrometer. Approximately 1–2 lg of purified
protein was electrophoresed on a 4–16% Bis–Tris gel. The Coomas-
sie-stained protein band was excised, reduced in-gel, alkylated, de-
stained, and proteolytically cleaved with trypsin following a
standard protocol [24]. Peptide mass maps were obtained by MAL-
DI-TOF mass spectrometry and the identity of the protein sample
was confirmed by a Mascot database search [25].
constants (Ki) were determined at KM level of the L-methionine
for each form of the MATs while varying the inhibitor concentra-
tions around their Ki values, and fitting the data by using a Dixon
plot [27].
The solution molecular weight of the recombinant MATs was
estimated by using dynamic light scattering (DLS) measurements.
Purified protein samples (1–2 mg/mL) were filtered using
a
0.1 m membrane filter for removal of particulates and then ana-
l
lyzed using a DynaPro Titan DLS (Wyatt Technology Corporation).
Data was acquired at 20 °C for 10 s, repeated 10 times and then
averaged. The Dynamics 6.7.3 software was used to fit the autocor-
relation function to extract the diffusion coefficient and hydrody-
namic radius from the Stokes–Einstein equation. The solution
molecular weight of the protein was estimated from the measured
hydrodynamic radius using the Rayleigh equation, and the oligo-
meric form of the enzyme was determined by dividing the solution
molecular weight with the monomer molecular weight.
Sequence alignment of AdoMet synthetases
Reference amino acid sequences for the studied Gram-negative
bacteria MATs were obtained from UniProt database: E. coli
(B1XFA4), P. aeruginosa (Q315Z0), N. meningitidis (E7BEH0), and
C. jejuni (A1W083). Sequences were aligned using the ClustalW
(version 2.0) program. Secondary structure alignment of the se-
quences with the E. coli structure (PDB ID: 1RG9) was constructed
using the ESPript program [28].
Native Gel electrophoresis of the purified AdoMet synthetases
were performed using the NativePAGE Novex 4–16% Bis–Tris gel
system. Approximately 20 lg protein samples were loaded onto
the gel and electrophoresed using light blue cathode buffer for
Results
Coomassie staining.
Enzyme expression, purification and characterization
Kinetic assay
The open reading frame of metK gene of each Gram-negative
organism, E. coli, P. aeruginosa, N. meningitidis and C. jejuni, was
cloned into a high copy plasmid for E. coli expression with a C-ter-
minal hexahistidine tag and an inducible T7 RNA polymerase pro-
moter. Pilot expression of the cloned metK gene of the MATs
showed that each of the recombinant enzymes was expressed at
37 °C. However, induction of protein expression at 28 °C allowed
proper folding of the proteins and increased enzyme activity levels
in the soluble fraction of the crude extracts.
Each recombinant enzyme of MAT was then purified to >95%
purity as described in Methods. After optimization, tens of milli-
grams of homogenous AdoMet synthetase were obtained from
5 g (wet weight) of cells expressing each of the recombinant
metK genes. SDS–PAGE shows a single band near the expected
size of 46–48 kDa for the different enzyme forms (Fig. 2A).
Mascot identification of the tryptic digest peptides of each of
the recombinant proteins confirmed the successful cloning and
identity of the AdoMet synthetases from each of the Gram-nega-
tive pathogens.
MAT activity was measured by a newly developed coupled en-
zyme assay that continuously monitors the production of pyro-
phosphate (Fig. 1). This assay avoids possible interference from
phosphate that is present in crude cell extracts. The reliability of
this assay was evaluated by comparing the kinetic parameters ob-
tained from the E. coli form of MAT against the published values ob-
tained from radioactive assays [1]. Routine assays were performed
at ambient temperature on a SpectraMax 190 plate reader by mon-
itoring the production of NADPH at 340 nm (
in an assay buffer containing 20 mM Tris–HCl, pH 7.6, 16 mM
MgCl2, 1 mM ATP, 0.1 mM
-methionine, 1 mM uridine-50-diphos-
phoglucose, 1 mM NADP, 10 M glucose-1,6-bisphosphate, 0.8
e )
= 6.22 mMꢀ1 cmꢀ1
L
l
units of glucose-6-phosphate dehydrogenase, 0.8 units of phospho-
glucomutase, and 0.1 units of uridine-50-diphosphoglucose
DLS studies carried out on each of the purified enzymes yielded
an approximate molecular weight of 170 kDa for most of the en-
zyme forms, confirming that these enzymes exist as a tetramer
in solution similar to the previously characterized E. coli enzyme.
The one exception is the C. jejuni enzyme, which yielded an
approximate molecular weight of 85 kDa by DLS, consistent with
a functional dimer in solution.
The oligomeric form of the MATs in solution was further con-
firmed by native gel electrophoresis. These enzyme forms pro-
duced a band in the range of 165–185 kDa, which is the expected
size for a tetrameric quaternary structure. The one exception is
the C. jejuni enzyme, with a band at about 85 kDa that is consistent
with a dimer in solution (Fig. 2B).
Fig. 1. Coupled AdoMet synthetase activity assay. The production of pyrophosphate
(PPi) is coupled with UDP-glucose (UDPG) and the resulting glucose 1-phosphate is
coupled to the production of NADPH after conversion to glucose 6-phosphate.

S.P. Zano et al. / Archives of Biochemistry and Biophysics 536 (2013) 64–71
67
Alternative substrates
To test our hypothesis that the members of this enzyme family
are capable of using alternative substrates to produce AdoMet
analogs, some derivatives of methionine (Fig. 3) were examined
to delineate the substrate specificity of the various MATs. D-Methi-
onine had previously been shown to be a substrate for MetK from
E. coli [29] and this unnatural isomer is also found to be a substrate
for the MATs from these pathogenic bacteria (Table 2). The kinetic
parameters were also determined for several carboxyl and amino
derivatives of
L-methionine that were found to function as
alternative substrates for these Gram-negative MATs (Table 2).
The N. meningitidis enzyme utilizes the methyl and ethyl esters
of L-methionine as alternative substrates with Vmax/Km values that
are only slightly diminished from that of the native methionine
substrate (Fig. 4, green bars). These methionine esters are also rea-
sonably good substrates for the E. coli enzyme, but with signifi-
cantly decreased catalytic efficiencies.
Additional substrate discrimination was found with these
methionine derivatives among the other MATs examined. These
same methionine esters are very poor substrates for the P. aerugin-
osa enzyme, with Vmax/Km values that are decreased by 10- to 100-
fold compared to the other related MAT forms (Fig. 4, red bars). The
methyl ester of methionine is a very poor substrate for the C. jejuni
enzyme and the ethyl ester is somewhat improved (Fig. 4, blue
bars). To examine the extent to which this substrate binding site
in the N. meningitidis and C. jejuni MATs can accommodate larger
methionine derivatives the t-butyl and phenyl esters of L-methio-
nine were synthesized and tested. The t-butyl ester is neither a
substrate nor an inhibitor of the MATs, while the phenyl ester is
a weak substrate for N. meningitidis MAT and shows about a 6-fold
improvement with the C. jejuni enzyme.
Fig. 2. Gel electrophoresis of the different forms of AdoMet synthetases. (A) SDS–
PAGE showing the relative sizes of the enzyme subunits. (B) NativePAGE showing
the quaternary structures of these enzyme forms.
Modifications at the amino group of methionine have a more
substantial impact on the capability of these derivatives to func-
tion as alternative substrates for these enzymes, and clearly show
discrimination between the C. jejuni and the other MAT orthologs.
Enzyme kinetics
The amino derivatives, N,N-dimethyl-L-methionine and N-acetyl-L-
The kinetic parameters of the recombinant MATs from each of
the Gram-negative organisms were determined by coupling the
PPi product through uridine-50-diphosphoglucose using a modified
uridine-50-diphosphoglucose pyrophosphorylase enzymatic assay
(Fig. 1). Table I reports the steady state kinetic parameters of each
of the purified MATs. The values obtained for the E. coli enzyme are
in good agreement with the previously published values [1], con-
firming the validity of the developed pyrophosphate based assay.
The Vmax value determined for the MAT from N. meningitidis is
nearly double, and the relative catalytic efficiency (Vmax/Km) of this
enzyme form for MgATP is more than twice that of the E. coli en-
zyme (Table 1). The P. aeruginosa enzyme has a Vmax value similar
to that of the E. coli MAT, but the relative catalytic efficiency for its
methionine, have the lowest activity among the identified alterna-
tive substrates for the family of MATs. In each case the Km value for
these amino derivatives has increased by a factor of 10 or higher
compared to the methionine esters (Table 2). The catalytic effi-
ciency (Vmax/Km) of N,N-dimethyl-
300- to 500-fold for the E. coli and N. meningitidis MATs compared
to -methionine, and was found to be an additional 20- to 30-fold
L-methionine is reduced by
L
lower for the P. aeruginosa enzyme. Furthermore, this compound is
not a substrate at all for the C. jejuni MAT. However, surprisingly,
the methyl ester derivative of this compound is a substrate for
the C. jejuni enzyme, albeit with a significantly higher Km value,
while this ester derivative did not show any substrate activity
with the P. aeruginosa, N. meningitidis or E. coli forms of MAT.
L-methionine substrate is nearly double while the value for MgATP
N-acetyl-L-methionine was also found to be a poor substrate for
is less than half that of the E. coli enzyme. In contrast to these rel-
atively modest changes, the MAT from C. jejuni is an outlier with a
5-fold higher catalytic turnover and a 3- to 4-fold higher catalytic
efficiency with each of the native substrates (Table 1).
the P. aeruginosa enzyme and a slightly better but still weak sub-
strate for the N. meningitidis MAT (Fig. 4). Some catalytic turnover
could be measured for the E. coli and C. jejuni enzyme forms, but it
Table 1
Kinetic parameters for AdoMet synthetases.
a
Enzyme form
Vmax
MgATP
L
-methionine
Km (mM)
Vmax/Km
Relative Vmax/Km
Km (mM)
Vmax/Km
Relative Vmax/Km
E. coli
1.22 0.12
2.01 0.17
1.17 0.25
6.43 0.40
0.070 0.001
0.110 0.011
0.040 0.005
0.090 0.002
1.7 ꢁ 104
1.8 ꢁ 104
2.9 ꢁ 104
7.1 ꢁ 104
1.0
1.1
1.7
4.2
0.120 0.008
0.080 0.001
0.30 0.02
1.0 ꢁ 104
2.5 ꢁ 104
3.9 ꢁ 103
3.1 ꢁ 104
1.0
2.5
0.4
3.1
N. meningitidis
P. aeruginosa
C. jejuni
0.21 0.02
a
lmol/min/mg of Protein.

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S.P. Zano et al. / Archives of Biochemistry and Biophysics 536 (2013) 64–71
molecular weight was obtained from dynamic light scattering
(DLS) studies that is consistent with a tetrameric quaternary struc-
ture, the same as was observed for the E. coli enzyme [30]. The one
exception is the MAT from C. jejuni, which is an apparent dimer in
solution. Similar observation was observed during native electro-
phoresis of the purified enzyme forms (Fig. 2B). These results place
the C. jejuni enzyme among the divergent class of MATs, which in-
cludes the archaeal enzyme from Methanococcus jannaschii and the
mammalian isoenzyme MAT III, each of which are dimers, and MAT
II, which is found in a dimer–tetramer equilibrium [31].
The structure of the E. coli MAT monomers are each identical,
but they use different surface interactions between the monomers
to form the functional tetramer, resulting in a dimer of dimers [30].
The interaction surface between each pair of dimers encompasses
about 1800 Å2 and incorporates several specific hydrogen bonding
and electrostatic interactions to maintain the tetrameric structure.
The amino acids that are involved in these specific subunit interac-
tions are altered in the C. jejuni enzyme, and these substitutions are
apparently sufficient to disrupt the dimer–dimer contacts. In the
E. coli MAT (PDB ID: 1RG9), the K97 from one dimer makes electro-
static interactions with D63 from the adjacent dimer and also with
E66 in the other domain of this adjacent dimer. This lysine at posi-
tion 97 has been replaced by a glutamate in the C. jejuni enzyme
and glutamate-66 with a glutamine. These substitutions would
eliminate the electrostatic attraction from the E. coli binding part-
ners across this dimer interface. Also, two pairs of serines (S80 and
S93) form complimentary hydrogen bonds across the dimer inter-
face, but in the C. jejuni enzyme these serines have been replaced
with a valine and a lysine, respectively. These changes in C. jejuni
MAT are apparently sufficient to alter its quaternary structure
and prevent association into a stable tetramer in solution.
Fig. 3. Chemical structures of the alternative AdoMet synthetase substrates.
was not possible to saturate either of these enzymes to measure a
Vmax value for this substrate.
Enzyme inhibitors
A few of the structural analogs of L-methionine tested against
the MAT did not support catalytic turnover, but instead were found
to be inhibitors of these enzymes. Screening of these inhibitors
against the different MATs showed very high selectivity against
these enzyme forms. S-Carbamyl-L-cysteine, a previously reported
modest inhibitor of the E. coli enzyme [1], showed a comparable Ki
value of about 4 mM against the P. aeruginosa enzyme. This inhib-
itor has a 3-fold decreased potency for the C. jejuni enzyme, but
showed no inhibition for the N. meningitidis enzyme when
examined at concentrations up to 40 mM. b-Methionine [(R)-3-
amino-5-(methylthio)pentanoic acid] was found to be an inhibitor
(Ki ꢂ 7 mM) of only the C. jejuni enzyme, with no inhibition ob-
served for the other AdoMet synthetases.
The mammalian AdoMet synthetase (MAT) from rat liver also
has alterations in the nature of the dimer interface interactions
that diminish substrate binding affinity and leads to a dimer–tetra-
mer equilibrium in solution [32]. In contrast to the ease of
dissociation of the MAT tetramer, the MetK from the archaeal ther-
mophile M. jannaschii is particularly stable to dissociation. The
overall thermal stability of this form of MetK has been ascribed
to this much tighter association of the dimers [33].
Discussion
Sequence alignment of the AdoMet synthetases
Oligomeric state of the AdoMet synthetases
An ESPript alignment [28] of the amino acid sequences of the
different forms of MATs shows 91% sequence identity between
our E. coli K12 strain and that of the E. coli F11 strain that had pre-
viously been structurally characterized [30]. The P. aeruginosa and
N. meningitidis enzymes also have high sequence identity to the
Purification of each of the recombinant MATs yielded a soluble
protein that gave a single band around 47 kDa upon SDS gel elec-
trophoresis. For most of the enzyme forms an apparent solution
Table 2
Methionine analog substrates for AdoMet synthetases.
Methionine Analogs
E. coli
P. aeruginosa
N. meningitidis
C. jejuni
a
a
a
a
Vmax
Km (mM)
Vmax
Km (mM)
Vmax
Km (mM)
Vmax
Km (mM)
1.22 0.12
0.43 0.03
0.81 0.11
n. d.
0.070 0.001
0.17 0.05
0.12 0.01
1.17 0.25
0.06 0.01
0.15 0.03
n. d.
0.040 0.005
0.62 0.09
0.65 0.09
2.01 0.17
2.88 0.04
3.02 0.02
0.08 0.02
0.65 0.11
0.14 0.09
0.11 0.01
0.24 0.01
0.18 0.02
0.91 0.33
9.8 0.2
6.4 0.4
0.26 0.03
5.0 0.1
0.09 0.002
0.52 0.06
0.53 0.04
1.3 0.2
L
L
L
L
L
-methionine
-methionine methyl ester
-methionine ethyl ester
-methionine phenyl ester
-ethionine
0.70 0.10
2.1 0.3
2.7 0.3
10.1 0.3
12.3 0.81
4.5 0.7
2.8 0.2
10.3 0.6
15.8 0.71
5.0 1.2
3.9 0.3
0.35 0.02
0.37 0.05
18.2 0.77
7.9 0.3
0.93 0.02
n. a.b
7.3 0.12
D
-methionine
N,N-dimethyl-
N,N-dimethyl-
N-acetyl-
L
L
-methionine
-methionine methyl ester
0.28 0.02
n. a.b
0.009 0.003
n. a.b
2.04 0.02
0.28 0.03
n. a.b
1.5 0..5
24.9 3.8
10.9 1.2
>20
c
c
L-methionine
–
>20
15.7 0.7
2.7 0.4
–
a
b
c
l
mol/min/mg of Protein.
No substrate activity with this enzyme form.
Very weak substrate.

S.P. Zano et al. / Archives of Biochemistry and Biophysics 536 (2013) 64–71
69
Fig. 4. Methionine analog specificity for AdoMet synthetases. The catalytic efficiencies (Vmax/Km) of the carboxyl and amino derivatives of methionine are compared for the
enzymes from E. coli (purple bars), P. aeruginosa (red bars), N. meningitidis (green bars) and C. jejuni (blue bars). The carboxyl derivatives are shown in solid bars and the less
active amino derivatives of L-methionine in striped bars, with the scale for the amino derivatives to the right.
AdoMet synthetase from this E. coli strain, 68% and 65%, respec-
tively. In contrast, the sequence of the MAT from C. jejuni has a
number of significant differences when compared with the se-
quence of this enzyme family (Fig. 5), with less than 40% overall se-
quence identity to the E. coli enzyme. Moreover, these alignments
also reveal the reported five amino acid conservation blocks (I–V)
(Fig. 5, black boxes) among the families of MATs [34], including
the methionine binding motifs (Fig. 5, asterisks), GHPDK, the ATP
binding motifs, GAGDQG, and a characteristic pyrophosphate rec-
ognition sequence for some MATs, DXGXTGRKII [35]. These same
inserts were also found in the MATs from all campylobacters and
related genera, including nitratiruptor and sulfurovum, but are not
found in the phylogenetically similar helicobacter MATs. In addi-
tion, the amino acid residues that play a direct role in substrate
binding in the E. coli enzyme (Fig. 5, triangles), and each of the res-
idues involved the binding of active site waters (Fig. 5, circles), are
conserved even in the low identity C. jejuni MAT. It is worth noting
that the E55 side chain of E. coli F11 MAT, which has been proposed
to participate in substrate binding through a hydrogen bond to the
consensus MAT sequence, with only 38% overall sequence identity
and five inserts of between three to six amino acids located
throughout the sequence (Fig. 5).
Substrate specificity of the AdoMet synthetases
L
-Methionine substrate analogs were screened to delineate the
substrate specificity of the MATs produced by each infectious
organism. The N. meningitidis and P. aeruginosa enzymes share high
sequence identity with the E. coli enzyme, and only small differ-
ences were observed in the catalytic efficiency for each native sub-
strate. By contrast, the low identity C. jejuni MAT has a 5-fold
higher catalytic turnover than the E. coli enzyme and 8-fold higher
catalytic efficiency with MgATP compared to the MetK from P.
aeruginosa (Table 1).
There are several previous reports of alternative methionine ana-
log substrates for the E. coli MAT, including L-ethionine and L-methi-
onine methyl ester [29], the S-vinyl and S-allyl analogs of
methionine [36], and 3,4-unsaturated methionine analogs [37].
amino group of
L
-methionine [30], is conserved among the en-
Screening of additional L-methionine derivatives revealed new
zymes from these Gram-negative pathogens (and among the vast
majority of E. coli strains), but has been replaced by an asparagine
in the MAT from the E. coli strain K12 used in this study. The kinetic
parameters for this E. coli MAT are quite similar to those reported
for this enzyme from the other E. coli strain, so presumably N55
participates in substrate binding in a similar fashion.
A secondary structure sequence alignment of the MATs exam-
ined in this study reveals that the few insertions and deletions in
the P. aeruginosa and N. meningitidis MATs fall outside of the regu-
lar secondary structural regions of the E. coli enzyme and are lo-
cated primarily in surface loops (Fig. 5). Conversely, many of the
highly conserved sequences among these orthologs are located in
the secondary structural elements that comprise the active site
structure and the subunit interface region. Therefore the structural
basis for the differences in substrate specificity and activity that
have been identified between these enzyme forms is likely due
to subtle and localized changes, and not a consequence of signifi-
cant structural rearrangements. In contrast, the sequence of the en-
zyme from C. jejuni has some significant differences from the
alternative substrates that can substitute for methionine in this
reaction with reasonably good catalytic activity (Table 2). Each of
the MATs studied show a preference for some of the carboxyl deriv-
atives that were tested. Several of these derivatives also showed
some selectivity among the different MAT orthologs. In contrast to
the lack of discrimination in catalytic efficiency for the natural sub-
strates, the N. meningitidis enzyme has a 2.5-fold increase in effi-
ciency with the ethyl and methyl esters of L-methionine relative to
the E. coli enzyme, while these esters are relatively poor substrates
for the P. aeruginosa enzyme with Vmax/Km values that are 25- to
30-fold lower. The C. jejuni enzyme uses the methyl ester with nearly
the same low efficiency as the P. aeruginosa enzyme, but shows
enhanced activity with the ethyl ester (Fig. 4).
This capability to catalyze amino acid activation with bulky es-
ter derivatives of methionine is presumably due to changes in the
L-methionine carboxylate binding site. Methionine is oriented at
the active site through an electrostatic interaction between K269
(E. coli MAT numbering) and the methionine carboxyl group, an
additional hydrogen bond to this group from Q98, and electrostatic

70
S.P. Zano et al. / Archives of Biochemistry and Biophysics 536 (2013) 64–71
Fig. 5. Sequence alignments of AdoMet synthetases from Escherichia coli (B1XFA4), Pseudomonas aeruginosa (Q315Z0), Neisseria meningitidis (E7BEH0), Campylobacter jejuni
(A1W083), Methanococcus jannaschii (DSM2661) and Rattus norvegicus (P13444). Black box: The five (roman numeral labeled) amino acid conservation blocks across all MATs;
Asterisks: methionine, ATP and pyrophosphate binding motif. The residues labeled with triangles are amino acids that directly bind the substrates and the circles indicate
amino acids surrounding bound waters that are involved in substrate binding, with the blue colored triangles and circles designating amino acids involved in the binding of
methionine.
L-

S.P. Zano et al. / Archives of Biochemistry and Biophysics 536 (2013) 64–71
71
bonds to the amino group from E55 and D236 [30]. Several active
References
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This work was supported by a grant (AI098702) from the Na-
tional Institutes of Health. We wish to thank Joshua Smith for
assistance with the purification of the AdoMet synthetases and
Dr. Robert Blumenthal (Department of Medical Microbiology and
Immunology, University of Toledo) for his insights and his critical
reading of this manuscript.