Functional asymmetry for the active sites of linked 5-aminolevulinate synthase and 8-amino-7-oxononanoate synthase

Article abstract of DOI:10.1016/j.abb.2011.05.002

5-Aminolevulinate synthase (ALAS) and 8-amino-7-oxononanoate synthase (AONS) are homodimeric members of the α-oxoamine synthase family of pyridoxal 5′-phosphate (PLP)-dependent enzymes. Previously, linking two ALAS subunits into a single polypeptide chain dimer yielded an enzyme (ALAS/ALAS) with a significantly greater turnover number than that of wild-type ALAS. To examine the contribution of each active site to the enzymatic activity of ALAS/ALAS, the catalytic lysine, which also covalently binds the PLP cofactor, was substituted with alanine in one of the active sites. Albeit the chemical rate for the pre-steady-state burst of ALA formation was identical in both active sites of ALAS/ALAS, the k_cat values of the variants differed significantly (4.4 ± 0.2 vs. 21.6 ± 0.7 min^-1) depending on which of the two active sites harbored the mutation. We propose that the functional asymmetry for the active sites of ALAS/ALAS stems from linking the enzyme subunits and the introduced intermolecular strain alters the protein conformational flexibility and rates of product release. Moreover, active site functional asymmetry extends to chimeric ALAS/AONS proteins, which while having a different oligomeric state, exhibit different rates of product release from the two ALAS and two AONS active sites due to the created intermolecular strain.

Full text of DOI:10.1016/j.abb.2011.05.002

Archives of Biochemistry and Biophysics 511 (2011) 107–117
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics
journal homepage: www.elsevier.com/locate/yabbi
Functional asymmetry for the active sites of linked 5-aminolevulinate synthase
and 8-amino-7-oxononanoate synthase
Tracy D. Turbeville a, Junshun Zhang a, W. Christopher Adams a, Gregory A. Hunter a, Gloria C. Ferreira a,b,c,
⇑
a Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, FL 33612, USA
b H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, FL 33612, USA
c Department of Chemistry, University of South Florida, Tampa, FL 33612, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
5-Aminolevulinate synthase (ALAS) and 8-amino-7-oxononanoate synthase (AONS) are homodimeric
members of the
-oxoamine synthase family of pyridoxal 50-phosphate (PLP)-dependent enzymes. Pre-
Received 15 March 2011
and in revised form 3 May 2011
Available online 11 May 2011
a
viously, linking two ALAS subunits into a single polypeptide chain dimer yielded an enzyme (ALAS/ALAS)
with a significantly greater turnover number than that of wild-type ALAS. To examine the contribution of
each active site to the enzymatic activity of ALAS/ALAS, the catalytic lysine, which also covalently binds
the PLP cofactor, was substituted with alanine in one of the active sites. Albeit the chemical rate for the
pre-steady-state burst of ALA formation was identical in both active sites of ALAS/ALAS, the kcat values of
the variants differed significantly (4.4 0.2 vs. 21.6 0.7 minꢀ1) depending on which of the two active
sites harbored the mutation. We propose that the functional asymmetry for the active sites of ALAS/ALAS
stems from linking the enzyme subunits and the introduced intermolecular strain alters the protein con-
formational flexibility and rates of product release. Moreover, active site functional asymmetry extends
to chimeric ALAS/AONS proteins, which while having a different oligomeric state, exhibit different rates
of product release from the two ALAS and two AONS active sites due to the created intermolecular strain.
Ó 2011 Elsevier Inc. All rights reserved.
Keywords:
5-Aminolevulinate
5-Aminolevulinate synthase
8-Amino-7-oxononanoate synthase
Pyridoxal 50-phosphate
a-Oxoamine synthase family
Introduction
structures of the a-oxoamine synthases are also highly conserved,
as demonstrated by the C root-mean-square deviation values for
SPT of 1.4–1.6 Å relative to the other three members [7]. Recently,
a
Pyridoxal 50-phosphate (PLP) is a necessary cofactor for a large
and catalytically versatile family of enzymes classified according
two new members, CqsA [14] and LqsA [15,16], joined the
a-oxo-
to structural and mechanistic homologies [1–5]. The
a-oxoamine
amine synthase subfamily. While both synthesize -hydroxyketone
a
synthases are a small but metabolically important subfamily within
fold-type I PLP-dependent enzymes and include 5-aminolevulinate
synthase (ALAS),1 8-amino-7-oxononanoate synthase (AONS), ser-
ine palmitoyltransferase (SPT), and 2-amino-3-oxobutyrate CoA li-
gase [6–13]. The catalytic cores of these four enzymes share
approximately 12% overall amino acid sequence identity, while the
identity between any pair is near 30% [10]. The three-dimensional
signaling molecules with major roles in quorum sensing [17], the
autoinducer synthase CqsA produces the cholera autoinducer-1
(CAI-1) [14] and the autoinducer synthase LqsA synthesizes the
Legionella autoinducer-1 (LAI-1) [15–17].
ALAS and AONS catalyze very similar reactions, namely the
Claisen-type condensation between a small amino acid and acyl-
CoA thioester, with concomitant decarboxylation of the amino
acid, leading to the formation of a 1–2-aminoketone product,
CoA, and carbon dioxide [3,10]. ALAS catalyzes the reaction be-
tween glycine and succinyl-CoA to give ALA, whereas AONS cata-
lyzes the mechanistically analogous reaction between alanine
and pimeloyl-CoA to yield 8-amino-7-oxononanoate (AON)
(Scheme 1). Both ALA and AON are essential metabolic com-
pounds: the first is the universal precursor to tetrapyrolles, [3,18]
and the second is an intermediate in biotin synthesis [19].
ALAS and AONS function as homodimers [8,10,18]. The active
sites are located at the subunit interface, where the PLP cofactor
is covalently bound to a conserved lysine residue through a Schiff
base linkage [8,10,20]. Each monomer consists of three domains, a
short N-terminal domain (ꢁ50 residues), a central catalytic domain
⇑
Corresponding author. Address: Department of Molecular Medicine, College of
Medicine, MDC 7, University of South Florida, 12901 Bruce B. Downs Blvd., Tampa,
FL 33612, USA. Fax: +1 813 974 0504.
E-mail address: gferreir@health.usf.edu (G.C. Ferreira).
Abbreviations used: ALAS, 5-aminolevulinate synthase; ALA, 5-aminolevulinate;
1
AONS, 8-amino-7-oxononanoate synthase; AON, 8-amino-7-oxononanoate; AMPSO,
3-([1,1-dimethyl-2-hydroxyethyl]amino)-2-hydroxypropane sulfonic acid; CD, circu-
lar dichroism; CoA, coenzyme A; DEAE, diethylaminoethyl; HEPES, N-(2-hydroxy-
ethyl) piperazine-N0-(2-ethane sulfonic acid); HPLC, high performance liquid
chromatography; MOPS, 4-morpholinepropanesulfonic acid; NAD+, b-nicotinamide
adenine dinucleotide; PCR, polymerase chain reaction; PLP, pyridoxal 50-phosphate;
PCoA, pimeloyl-coenzyme A; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel
electrophoresis; SPT, serine palmitoyltransferase; SCoA, succinyl-coenzyme A; WT,
wild-type.
0003-9861/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.abb.2011.05.002

108
T.D. Turbeville et al. / Archives of Biochemistry and Biophysics 511 (2011) 107–117
(ꢁ250 residues), and
a
C-terminal domain (ꢁ100 residues).
Although all three domains participate in dimerization, the cata-
lytic domain contributes the most to the dimeric interface
[7,8,10]. Shared active sites at the intersubunit interface of dimeric
proteins elicit major questions about the roles of residues from
either subunit toward the activity of the enzyme, cooperativity of
the active sites, and contributions of intersubunit interactions to
any one active site.
Scheme 1.
The highly conserved tertiary structure of
a-oxoamine syn-
thases can allow for substantial plasticity, as demonstrated with
circularly permuted murine erythroid ALAS variants [21,22]. Rear-
rangement of some secondary structural elements of ALAS does
not prevent folding of the polypeptide chain into a structure com-
patible with binding of the PLP cofactor and assembly of the two
subunits into a functional homodimer [21,22]. Simply linking the
two ALAS monomers with a two amino acid spacer results in a
monomeric enzyme (or ‘‘single chain dimer’’) with distinct spec-
troscopic properties and substantially enhanced enzymatic activity
compared to homodimeric ALAS [23]. Similarly, a naturally occur-
ring marine viral ‘‘single chain dimeric’’ SPT has been discovered,
and construction of yeast and mammalian SPT single-chain chime-
ras formed by linking the two highly similar, but structurally un-
ique, LCB1 and LCB2 subunits resulted in functional enzymes, but
with altered substrate specificities [24]. In addition, Han et al.
[25] recently reported the finding of two small subunits of mam-
malian SPT (ssSPTa and ssSPTb), which can confer distinct acyl-
CoA substrate specificities to the enzyme.
To investigate the unusual enhanced enzymatic activity result-
ing from linking ALAS dimers to form a monomer and help define
the limits of structural plasticity in a-oxoamine synthases, we con-
structed and characterized ‘‘single chain dimeric’’ ALAS variants, in
which one of the two active sites harbored a mutation eliminating
measurable enzyme activity, and single-chain chimeras between
WT or mutated ALAS and AONS. By combining two subunits into
a single polypeptide chain, we altered one active site, while leaving
the other intact, and hence assessed the role of each active site in
the ALAS dimer. We report that the two active sites in ALAS/ALAS
differentially contribute to the enhanced activity of the enzyme,
even though the amount of ALA produced during the first turnover
is identical in both active sites. Further, the chimeric ALAS/AONS
protein forms multi-functional dimers with both ALAS and AONS
activities.
Materials and methods
Materials
Fig. 1. ALAS and AONS dimmers. (A and B) ALAS and AONS homodimers in ribbon
representation with one subunit shown in yellow and the central catalytic core, N-
terminal domain, and C-terminal domain of the second subunit rendered in dark,
medium and light blue, respectively. (A) ALAS homodimer from R. capsulatus (PDB
code: 2BWN). The PLP cofactor (red), the active site lysine (green) involved in the
Schiff base linkage with PLP (K248 in R. capsulatus ALAS and K313 in murine
erythroid ALAS) and F276 (purple; F341 in murine erythroid ALAS) are depicted in
ball-and-stick representation. (B) AONS homodimer from E. coli (PDB code: 1BS0).
The PLP cofactor (red) and the active site lysine (green) involved in the Schiff base
linkage with PLP (K236) are depicted in ball-and-stick representation. (C) Schematic
representation of the expression plasmids for mutated ALAS, AONS, ALAS/ALAS
variants, and single-chain chimeras between ALAS (WT or mutated) and AONS (WT
or mutated). Each of the expression plasmids contains a DNA fragment, encoding
either a single protein subunit or a chimeric protein, under the control of the phoA
promoter [26] and possesses an ampicillin resistance selectable marker (see
‘‘Materials and methods’’ for details). Ampr, ampicillin resistance gene; Bam HI, Mfe I
and Sal I, cloning sites.
All restriction enzymes, Vent DNA polymerase, and T4 DNA li-
gase were from New England Biolabs. Superdex 200 gel filtration
resin was from Amersham Biosciences-GE Healthcare and DNA oli-
gonucleotides were from Integrated DNA Technologies. Sodium
dodecyl sulfate polyacrylamide gel electrophoresis reagents were
acquired from Bio-Rad. All other reagents were purchased from
Sigma–Aldrich Chemical Company or Fisher Scientific.
Methods
Construction of ALAS/ALAS and ALAS/AONS chimeric expression
plasmids
Chimeras between murine, mature erythroid ALAS and Esche-
richia coli AONS or between mutated forms of either ALAS or AONS
and the WT enzymes were engineered using pGF23 [26] as the
expression vector (Fig. 1, Table 1). pTDT1, an expression plasmid
for 6ꢂ-histidine-tagged E. coli AONS, was constructed by PCR-
amplification of the E. coli bioF gene, which encodes the AONS pro-
tein, and subcloning of the PCR product into pGF23, such that the
bioF gene replaced the ALAS-encoding fragment (see Supplemen-
tary data).

T.D. Turbeville et al. / Archives of Biochemistry and Biophysics 511 (2011) 107–117
109
Table 1
Nomenclature defining the plasmids and enzymes described in this report.
Plasmid
Proteina
Description
Massb (kD)
Reference
pGF23
pTDT1
pGF27
pTDT8
pAC1
ALAS
ALAS homodimer
AONS homodimer
56
40
56
56
112
112
112
96
96
96
[26]
This study
[27,37]
This study
[23]
This study
This study
This study
This study
This study
This study
This study
This study
This study
AONS
ALASK313A
ALAS homodimer harboring the K313A mutation
ALAS homodimer harboring the F341A mutation
Single polypeptide ALAS homodimer
Chimera of ALASK313A and ALAS
Chimera of ALAS and ALASK313A
Chimera of ALAS and AONS
ALASF341A
ALAS/ALAS
pCA1
ALASK313A/ALAS
ALAS/ALASK313A
ALAS/AONS
AONS/ALAS
ALASK313A/AONS
AONS/ALASK313A
ALASF341A/AONS
AONS/ALASF341A
ALAS/AONSK236A
pMG1
pTDT5
pTDT4
pTDT12
pTDT17
pTDT14
pTDT15
pTDT7
Chimera of AONS and ALAS
Chimera of ALASK313A and AONS
Chimera of AONS and ALASK313A
Chimera of ALASF341A and AONS
Chimera of AONS and ALASF341A
Chimera of ALAS and AONSK236A
96
96
96
96
a
In the linked proteins the first abbreviation refers to the N-terminal enzyme.
Monomeric molecular mass.
b
The pTDT5 and pTDT4 expression plasmids (Table 1) were con-
structed to yield chimeric proteins between ALAS and AONS
(Fig. 1). Using the 50–30 convention for the ‘‘chimeric gene’’ under
the control of the alkaline phosphatase promoter, pTDT5 contains
the cDNA coding for ALAS linked to the following bioF gene for
AONS through a Mfe I site, whereas in pTDT4, the bioF gene pre-
cedes the ALAS cDNA (Fig. 1). These constructs were based on
the pAC1 plasmid [21–23] (Fig. 1), which contains two tandem
ALAS cDNA sequences separated by an MfeI cloning site.
The pTDT12 and pTDT17 expression plasmids (Table 1) were
constructed using the pGF27 expression plasmid for the ALASK313A
variant [27] as the starting material for a DNA piece coding for the
ALASK313A mutation. The pGF27 plasmid was digested with Kpn I
and Xba I, and the ALASK313A-encoding fragment was ligated into
pTDT4 and pTDT5 digested with the same enzymes.
The pTDT8 plasmid (Table 1) was designed to encode a full-
length ALAS with phenylalanine-341 mutated to alanine. To intro-
duce the F341A encoding mutation into ALAS cDNA, the method
previously described by Gong et al. was followed [28].
The pTDT14 and pTDT15 expression plasmids (Table 1) were
constructed using pTDT8 as the source plasmid for the ALAS-
F341A-encoding fragment. The pTDT8 plasmid was digested with
Kpn I and Xba I, and the fragment containing the ALASF341A muta-
tion was isolated and subcloned into pTDT5 and pTDT4 to generate
pTDT14 and pTDT15, respectively.
1 L), 2 mM MgSO4, 0.1 mM CaCl2, 0.4% glucose, 0.1% vitamin-free
casamino acids and 1.5% agar.
Purification of ALAS, AONS, ALAS/ALAS, ALASK313A/ALAS, ALAS/
ALASK313A and ALAS/AONS
The purification of ALAS from E. coli DH5
pGF23 was as previously described [26]. Recombinant E. coli AONS
and chimeric ALAS/AONS were purified from E. coli DH5 cells har-
a cells harboring
a
boring pTDT1 and pTDT5, respectively. Tandem ALAS variants (i.e.,
ALAS/ALAS, ALASK313A/ALAS and ALAS/ALASK313A) were purified
from E. coli strain BL21(DE3) overproducing cells harboring pAC1,
pCA1 or pMG1. ALAS, AONS and the above tandem variants were
expressed and purified following slight modifications to the purifi-
cation described in [26]. Specifically, for both AONS and ALAS/
AONS, the initial ammonium sulfate fractionation step was 20%.
After stirring for 20 min at 4 °C, the solution was centrifuged at
27,000g for 30 min at 4 °C, and the supernatant was further frac-
tionated with ammonium sulfate to a final concentration of 40%.
For the purification of AONS, the protein pellet was resus-
pended in buffer A and loaded onto an Ultrogel ACA-44 gel filtra-
tion column equilibrated with buffer A. The protein solution was
adjusted to 20% (w/v) ammonium sulfate and the subsequent chro-
matographic steps using Phenyl-Sepharose and Q-Sepharose anion
exchange columns were as previously described [29].
The purification of ALAS/ALAS from bacterial cells harboring the
pAC1 expression plasmid (Table 1) was according to a previously
published method [21–23]. For the tandem ALAS variants, ALAS-
K313A/ALAS and ALASK313A/ALAS encoded by pCA1 and pMG1,
respectively (Table 1), the protein pellet was resuspended in buffer
A and loaded onto a Superdex 200 column equilibrated with buffer
A. The fractions containing protein were pooled and loaded onto a
DEAE-sephacel resin equilibrated with buffer A. The resin was
washed with buffer A, and the protein was eluted with buffer A
containing 70 mM KCl. For the purification of ALAS/AONS, the pro-
tein pellet obtained after 40% ammonium sulfate fractionation was
resuspended in buffer A, pH 7.9, and loaded onto an Ultrogel ACA-
44 column equilibrated with the same buffer. The fractions con-
taining protein were pooled and loaded onto a Q-Sepharose col-
umn equilibrated with buffer A, pH 7.9. The Q-Sepharose resin
was washed with buffer A, pH 7.9, containing 25 mM KCl, until
Abs280 of the ‘‘washed proteins’’ was lower than 0.1; ALAS/AONS
was eluted from the Q-Sepharose resin with a 100–150 mM KCl
gradient in buffer A, pH 7.5. The protein purity was assessed using
SDS–PAGE. When the final product did not meet our purity criteria
(i.e., over 95% homogeneity as judged by SDS–PAGE), size exclusion
The pTDT7 expression plasmid encodes the ALAS/AONS chimera
in which the AONS active-site lysine involved in the Schiff base
linkage with the PLP cofactor, K236, is mutated to an alanine (Table
1; Fig. 1). The method described by Gong et al. [28] was used to
introduce the K236A-encoding mutation in the bioF gene harbored
in pTDT5.
The pCA1 and pMG1 expression plasmids, encoding ALASK313A
/
ALAS and ALAS/ALASK313A, respectively, were constructed using the
pAC1 and pGF27 plasmids (Table 1; Fig. 1).
Biological screening for ALAS and AONS function
Competent E. coli HU227 and R872 cells were transformed by
electroporation with expression plasmids containing the ALAS
and AONS chimeric constructs. To screen for ALAS function, trans-
formed HU227 cells were plated on Luria–Bertani medium (0.5%
yeast extract, 1% tryptone, 1.0% NaCl and 1.5% agar) containing
50
R872 cells were plated on M9 minimal medium containing
50
g/ml ampicillin. M9 medium contains 1ꢂ M9-salts (12.8 g
Na2HPO4–7H2O, 3.0 g KH2PO4, 0.5 g NaCl, and 1.0 g NH4Cl per
lg/ml ampicillin. To screen for AONS function, transformed
l

110
T.D. Turbeville et al. / Archives of Biochemistry and Biophysics 511 (2011) 107–117
chromatography with Sephadex 200 resin was used to eliminate
protein contaminants. Protein-containing fractions were pooled
and concentrated in an Amicon 8050 stirred cell with an YM30
membrane. The purified and concentrated enzyme (WT ALAS,
AONS, ALAS/ALAS, ALASK313A/ALAS, ALAS/ALASK313A or ALAS/AONS)
was stored in liquid nitrogen.
program SigmaPlot, where Pt represents the product concentration
at an aging time t, A is the amplitude of the burst phase, kb is the
burst rate constant, kss is the steady-state rate constant, and E0 is
the total enzyme concentration [33].
bt
Pt ¼ Að1 ꢀ eꢀk Þ þ kssE0t
ð1Þ
Protein concentrations were determined with the bicinchoninic
acid method using bovine serum albumin as the standard [30].
ALAS/AONS concentrations are reported on the basis of a subunit
Results
molecular mass of 96 kD, while ALAS/ALASK313A and ALASK313A
/
In vivo activity screen of ALASK313A/ALAS and ALAS/ALASK313A
ALAS concentrations are reported on the basis of a subunit molec-
ular mass of 112 kD.
Positive genetic complementation of hemAꢀ E. coli HU227 cells
was utilized to assess the ALAS activity of ALASK313A/ALAS and
ALAS/ALASK313A, which harbor the inactivating K313A mutation
in either of the two linked ALAS subunits (Table 1 and Fig. 1).
HU227 cells cannot produce ALA and only grow in a medium sup-
plemented with ALA [35] or when transformed with a functional
ALAS expression plasmid [18,36]. Indeed, while HU227 cells har-
boring the ALAS homodimer or the single chain dimeric ALAS/ALAS
can grow in a medium without ALA [18,36], HU227 cells overpro-
ducing the K313A homodimer cannot [18] (Table 2). Both ALAS-
K313A/ALAS and ALAS/ALASK313A variants retained function as
indicated by the ability of transformed HU227 cells to grow when
harboring either variant (Table 2).
Molecular mass determination by gel filtration chromatography
The native molecular mass of ALAS/AONS was determined using
gel filtration chromatography as previously described by Cheltsov
et al. [21].
Pimeloyl-CoA synthesis
Pimeloyl-CoA was synthesized as previously described by Ploux
and Marquet [19]. Purity was assessed by reverse-phase HPLC and
concentration was determined by measuring the absorbance at
260 nm and using an
e .
260nm = 16,800 Mꢀ1 cmꢀ1
Spectroscopic measurements
Absorption spectra were acquired at ambient temperature
using a Shimadzu UV 2100 dual beam spectrophotometer, with a
reference containing all components except the purified enzyme.
Fluorescence spectra were collected on a Shimadzu RE-5301 PC
Spectroscopic characterization of ALASK313A/ALAS and ALAS/ALASK313A
Although, as previously reported [23], the UV/visible absor-
bance spectra for both ALAS and the single chain dimeric ALAS/
ALAS exhibited the characteristic maxima for PLP-dependent en-
zymes at ꢁ330 and ꢁ420 nm, the prominence of these absorbance
bands varied between the spectra of the two proteins (Fig. 2). At pH
7.5 the ratio of the 420–330 nm absorbance changed from 0.44 for
ALAS to 0.67 for ALAS/ALAS (Fig. 2). Similarly, the distinct absor-
bances at these wavelengths in the UV/visible spectra of the ALAS-
K313A/ALAS and ALAS/ALASK313A variants resulted in a 420–330 nm
absorption ratio of 0.35 and 0.58 at pH 7.5 for ALASK313A/ALAS and
ALAS/ALASK313A, respectively (Fig. 2). The ꢁ330 and ꢁ420 nm max-
ima were previously attributed to the substituted aldimine and
ketoenamine forms of the internal aldimine between PLP and the
spectrofluorophotometer using protein concentrations of 2–4 lM.
Fluorescence blank spectra were collected from samples contain-
ing all components except enzyme immediately prior to the mea-
surement of samples containing enzyme. The blank spectra were
subtracted from the spectra of samples containing enzyme.
Steady-state kinetic characterization of ALAS/AONS, ALASK313A/ALAS,
and ALAS/ALASK313A
ALAS steady-state activity of the ALAS/AONS chimera, ALAS-
K313A/ALAS and ALAS/ALASK313A was determined at 30 °C using a
continuous spectrophotometric assay as described previously for
WT ALAS [31]. AONS steady-state activity of the of the ALAS/AONS
chimera was determined at 30 °C using a coupled enzymatic spec-
trophotometric assay for AONS [32], which is similar to that devel-
oped for determination of ALAS activity [31]. Briefly, in the latter
e-amino group of K313, respectively [23]. Clearly, the lysine muta-
tions reveal that the cofactor internal aldimines differ between the
two active sites in the linked enzymes.
assay,
a-ketoglutarate dehydrogenase was replaced by pyruvate
dehydrogenase as the coupling enzyme, and the reactions con-
Kinetic characterization of the ALASK313A/ALAS and ALAS/ALASK313A
variants and determination of the dissociation constants for the
binding of ALA
tained 20 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM pyruvic acid,
1 mM NAD+ and 0.25–1
lM enzyme. Data were acquired using a
Shimadzu UV 2100 dual-beam spectrophotometer. Enzymatic
activity data were plotted vs. substrate concentration in which
one of the substrate concentrations varied, while the second was
The steady-state kinetics of the ALASK313A/ALAS and the ALAS/
ALASK313A reactions were examined, and the results are presented
in Table 3. Previously, it was reported that linking the two ALAS
subunits into the single chain dimeric ALAS/ALAS resulted in over
5-fold and 28-fold increases in the kcat and KSmCoA values, respec-
tively [23]. To determine the individual contribution of the two ac-
tive sites to the overall steady-state activity of ALAS/ALAS, the
steady-state kinetic parameters of the ALASK313A/ALAS and ALAS/
ALASK313A variants, in which the K313A mutation [27,37] was inde-
pendently introduced in each of the two active sites, were deter-
mined (Table 3). While the K313A mutation in ALASK313A/ALAS
decreased the kcat 2.5-fold, the same mutation in ALAS/ALASK313A
resulted in a 12.6-fold decrease of the kcat value. Similarly, the cat-
alytic efficiencies of ALASK313A/ALAS for glycine and succinyl-CoA
were decreased approximately 2.9- and 1.8-fold, respectively,
whereas those of ALAS/ALASK313A were reduced approximately 9-
and 51-fold for glycine and succinyl-CoA, respectively. These
kept constant. The steady-state kinetic parameters (i.e. , KmGly
,
KSmCoA, and kcat of the ALAS/AONS chimera, ALASK313A/ALAS and
ALAS/ALASK313A and KmAlanine, KmPCoA, and kcat of the ALAS/AONS chi-
mera) were determined by fitting the data to the Michaelis–Men-
ten equation using non-linear regression analysis software.
Rapid chemical quenched-flow experiments and data analysis
Rapid chemical quenched-flow experiments were performed
using a SFM-400/Q mode quenched-flow apparatus (BioLogic Sci-
ence Instruments, France), equipped with a circulating water bath
to control the temperature of the reactants as described in Zhang
and Ferreira [33]. ALA concentration in the quenched samples
was also determined as previously described [33]. ALA produced
at different reaction times were plotted against time and fitted to
Eq. (1) [34], using the nonlinear least-squares regression analysis

T.D. Turbeville et al. / Archives of Biochemistry and Biophysics 511 (2011) 107–117
111
Table 2
Growth of transformed E. coli strains on selective media.
Plasmid
Protein
E. coli HU227
E. coli R872
LB/Amp/ALA
LB/Amp
LB/Amp
Biotinꢀ minimal
pGF23
pTDT1
pGF27
pTDT8
pAC1
ALAS
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
ꢀ
+
AONS
ꢀ
ꢀ
ꢀ
+
+
+
ALASK313A
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
+
+
+
+
ALASF341A
ALAS/ALAS
pCA1
ALASK313A/ALAS
ALAS/ALASK313A
ALAS/AONS
AONS/ALAS
ALASK313A/AONS
AONS/ALASK313A
ALASF341A/AONS
AONS/ALASF341A
ALAS/AONSK236A
pMG1
pTDT5
pTDT4
pTDT12
pTDT17
pTDT14
pTDT15
pTDT7
+
+
ꢀ
ꢀ
ꢀ
ꢀ
+
+
ꢀ
leading to ALA release [23]. To determine if the rate-limiting step
of the reactions catalyzed by the K313A single chain dimeric vari-
ants occurs after the chemical step, we explored the events occur-
ring at the active sites by performing rapid quenched-flow
experiments and seeking a pre-steady-state burst of product for-
mation in the ALASK313A/ALAS- and ALAS/ALASK313A-catalyzed
reactions (Fig. 3). The time courses for the reaction of either ALAS-
K313A/ALAS or ALAS/ALASK313A were determined by mixing a solu-
tion containing glycine (400 mM)-saturated enzyme (30
lM)
with succinyl-CoA (300 M). At various times after mixing, the
l
reaction was quenched by the addition of 0.14 M perchloric acid
(final concentration) and the ALA concentration in the quenched
reaction mixture was determined as described in [33]. The time-
dependent formation of ALA is illustrated in Fig. 3. The reaction
of ALASK313A/ALAS occurred with a burst in ALA production at a
rate of 30.6 4.2 sꢀ1, whereas the ALA formation burst of the
ALAS/ALASK313A reaction was at a rate of 45.1 6.1 sꢀ1. These burst
rates are similar to the burst rate previously reported for the ALAS/
ALAS reaction, 48.6 6.1 sꢀ1 [23]. Further, in both cases, the ALA
burst was followed by a steady-state product accumulation at a
significantly slower rate (<100-fold), corresponding to a rate-limit-
ing step preceding subsequent turnover. We assigned this step to
ALA release, presumably a protein conformational change leading
to ALA release as postulated for the ALAS- and ALAS/ALAS-cata-
lyzed reactions [33,38]. Another important piece of information
provided by the burst experiments shown in Fig. 3 relates to the
burst amplitudes, representing the concentration of ALAS active
sites for the reactions. The burst amplitudes were 0.25/active site
Fig. 2. Absorption spectra of ALAS, ALAS/ALAS, ALASK313A/ALAS and ALAS/
ALASK313A. The inset includes the region from 300 to 500 nm. Protein concentrations
were adjusted to 7.75 lM for ALAS/ALAS, 4.25 l
M for ALASK313A/ALAS and ALAS/
ALASK313A or 10 for ALAS in 20 mM Hepes, pH 7.5. ALAS/ALASK313A (—),
lM
ALASK313A/ALAS (d d d), ALAS/ALAS (- -), and ALAS (– –).
findings suggest that linking the N-terminus of one subunit to the
C-terminus of the other subunit in ALAS/ALAS may have created
strain that slightly hindered the steady-state enzymatic activity
of the N-terminal active site, while accentuating the activity of
the C-terminal active site.
and 0.21/active site for ALASK313A/ALAS and ALAS/ALASK313A
,
respectively, nearly 50% of the burst amplitude previously deter-
mined for ALAS/ALAS, 0.49/active site [23]. These results are con-
Pre-steady-state burst experiments for ALASK313A/ALAS and ALAS/
ALASK313A
sistent with the number of the active sites of either ALASK313A
/
ALAS or ALAS/ALASK313A being roughly 50% of that of ALAS/ALAS
and also indicate that the different contributions of each of the
two active sites of ALAS/ALAS to its steady-state activity emanate
from a step occurring after ALAS chemistry.
As with the ALAS-catalyzed reaction, the rate-limiting step for
the single chain dimeric ALAS occurs after the chemical step and
is proposed to be associated with a protein conformational change
Table 3
Summary of steady-state kinetic parameters for ALAS, ALAS/ALAS, ALAS/ALASK313A and ALASK313A/ALAS.
kcat (minꢀ1
)
KGmly (mM)
kcat/KmGly (minꢀ1 mMꢀ1
)
KSmCoA
(lM)
kcat/KmSCoA (minꢀ1
l
Mꢀ1
)
KAdLA
(lM)
ALAS
10 1a
55.4 0.1b
4.4 0.2
23 1a
0.43 0.06a
3.32 0.04b
0.37 0.09
1.46 0.17
2.3 0.1a
0.45 0.03b
1.8 0.3
4.3 0.2a
123 8b
2.4 0.4
25 3a
1.46 0.01
6.8 1.1
2.3 0.8
ALAS/ALAS
16.7 0.2b
11.8 2.8
14.8 11.7
ALAS/ALASK313A
ALASK313A/ALAS
21.6 0.7
0.32 0.05
67.5 10.8
a
Data from Ref. [28].
Data from Ref. [23].
b

112
T.D. Turbeville et al. / Archives of Biochemistry and Biophysics 511 (2011) 107–117
Fig. 4. Schematic representation illustrating the active site arrangement for ALAS/
ALAS and possible active site arrangements for ALAS/AONS. Red bars represent
ALAS polypeptide chains, with the essential active site residues K313 and F341 from
one single polypeptide chain contributing to separate active sites. Green bars
represent AONS polypeptide chains, with the essential active site residues K236 and
Y264 from one single polypeptide chain contributing to separate active sites. In (A)
and (B), there are two active sites per single chain dimer, whereas in (C) and (D),
there are four active sites per chimeric ALAS/AONS dimer. (A) Active site
arrangement of the ALAS/ALAS ‘‘single chain dimer’’. (B)–(D) Possible active site
arrangements for ALAS/AONS. (B) ALAS/AONS single chain dimer. (C) ALAS/AONS
chimeric dimer with ‘‘hybrid’’ active sites comprised of both ALAS and AONS
residues. (D) ALAS/AONS chimeric dimer with two of the active sites containing
only ALAS residues and the other two active sites containing only AONS residues.
(For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)
Fig. 3. Kinetics of a pre-steady-state burst of ALA product in the ALAS/ALASK313A
and ALASK313A/ALAS reactions. ALAS/ALASK313A (square) or ALASK313A/ALAS (trian-
gle) (15
lM) preincubated with glycine (200 mM) was quickly reacted with
succinyl-CoA (150
lM) at 20 °C. The concentrations shown in parentheses are final
concentrations after mixing. The inset illustrates the first 0.3 s of the reaction time
course. The curves represent the best fits to Eq. (1). For comparison, the time course
for the reaction of ALAS/ALAS (8.6 lM) (filled circles) under the experimental
conditions described above is included [23].
Biological screening for ALAS and AONS function
The determination of the crystallographic structures of ALAS [8]
and AONS [10] confirmed the prediction drawn from the high degree
of sequence similarity between ALAS and AONS (37%) that these two
critical for function of the murine erythroid ALAS homodimer, of
the corresponding K236 and Y264 [10], only K236 is essential for
function in the E. coli AONS homodimer.
a
-oxoamine synthases have similar 3D fold and active site architec-
ture. To extend our studies on the plasticity of the PLP-binding and
active site of ALAS to other members of the -oxoamine synthase
The three possible arrangements for the active sites of ALAS/
AONS chimera are (1) formation of two active sites with the contri-
bution of amino acids from one ALAS and one AONS polypeptide
chains to each active site (Fig. 4B) in an analogous arrangement
to that of single chain dimeric ALAS/ALAS (Fig. 4A); (2) formation
of four active sites with the contribution of amino acids from one
ALAS and one AONS polypeptide chains to each active site
(Fig. 4C); (3) formation of four active sites with two of the active
sites being formed with only ALAS amino acids and the other
two of the active sites with only AONS amino acids (Fig. 4D), such
that they represent WT ALAS- and AONS-like active sites. While the
first active site arrangement would result from one single, chimeric
polypeptide chain (Fig. 4B), the latter two active site arrangements
would arise from two chimeric polypeptide chains (Fig. 4C and D).
The determined molecular mass of ꢁ182 KDa for the chimeric
ALAS/AONS (see below and Fig. 5) ruled against a single chain, di-
meric ALAS/AONS and the active site arrangement depicted in
Fig. 4B. To distinguish between the other two possibilities for the
active site arrangement of the ALAS/AONS chimera (Fig. 4C and
D), an experimental approach involving the use of specific amino
acid mutations targeted to abolish either ALAS or AONS function
and biological selection systems (E. coli HU227 and E. coli R872)
was followed. If the active site arrangement in Fig. 4C were correct
the K313A mutation would eliminate ALAS function in two of the
four active sites, and the AONS residues in one or two of the
remaining sites would have to complement the ALAS residues,
yielding a chimeric protein with ALAS and AONS activities. In con-
trast, with the active site arrangement presented in Fig. 4D, the
ALAS and AONS activities arise from WT ALAS- and AONS-like ac-
tive sites, and thus a deleterious mutation of a critical active site
residue in ALAS or AONS would produce a chimeric enzyme with
impaired ALAS or AONS activity. HU227 cells transformed with
either pTDT12 or pTDT17 [i.e., expression plasmids for the ALAS/
a
family, chimeras of ALAS and AONS and chimeras of singly-mutated
ALAS variants and AONS were constructed (Table 1 and Fig. 1). The
two major objectives were (1) to determine whether the chimeras
retain ALAS and AONS activities and (2) to examine the active site
arrangement in the ALAS/AONS chimera. Both ALAS/AONS and
AONS/ALAS exhibited ALAS and AONS activities as assessed by the
positive genetic complementation of hemAꢀ HU227 and bioFꢀ
R872 cells, which are ALA/heme [35,39] and biotin auxotrophs
[40], respectively (Table 2). From these in vivo activity assays,
ALAS/AONS and AONS/ALAS appear to be bifunctional enzymes.
To start addressing the question related to the active site
arrangement of ALAS/AONS chimera, directed-mutagenesis of crit-
ical residues in ALAS and AONS had to be established. K236 of
E. coli AONS, the conserved active site lysine involved in PLP bind-
ing and catalysis [32], corresponds to K313 of murine ALAS [3,37].
Thus, when the K236A mutation was introduced in the AONS
homodimer, bioFꢀ R872 cells did not grow in a medium without
biotin (data not shown), similar to the absence of growth of hemAꢀ
HU227 cells overproducing ALASK313A when plated onto a medium
lacking ALA (Table 2). The crystal structure of the Rhodobacter cap-
sulatus ALAS homodimer revealed that amino acids of the two
polypeptide chains contribute to the same active site, which is lo-
cated at the dimer interface [8]. For example, F276 (R. capsulatus
ALAS numbering or F341 in the equivalent murine erythroid ALAS
numbering), a phenylalanine crucial for interaction with the pante-
theine moiety of CoA, and the lysine involved in the Schiff base
linkage participate in the architecture of the same active site but
reside in different polypeptide chains. Of significance to this study,
F341 is critical to ALAS function, as the F341A mutation abolished
the production of ALA necessary to sustain growth of HU227 cells
on a selective medium (Table 2). Thus, while K313 and F341 are

T.D. Turbeville et al. / Archives of Biochemistry and Biophysics 511 (2011) 107–117
113
Fig. 5. Determination of the molecular mass of the ALAS/AONS chimera by gel
filtration chromatography. ALAS/AONS (5 mg) was applied to a Pharmacia Sephadex
200 filtration column and eluted as previously described [21]. The molecular mass
calibration curve for the Superdex 200 column used cytochrome c (12.4 kDa),
carbonic anhydrase (29.0 kDa), ovalbumin (45 kDa), bovine serum albumin
(66.0 kDa), alcohol dehydrogenase (150.0 kDa), and b-amylase (200.0 kDa) as
protein standards (indicated by circles). The ALAS/AONS chimera is indicated by a
triangle. (Inset) 12.5% SDS–PAGE of the purified ALAS, AONS and ALAS/AONS
chimera, which were detected using Coomassie Brilliant Blue staining. Approxi-
mately 5 lg of each protein sample was loaded per lane.
AONS chimera harboring the K313A mutation in ALAS (i.e., Table
1)] did not support the growth of these cells in an ALA-depleted
medium (Table 2). However, R872 cells harboring either of these
two plasmids could grow in a medium without biotin. A similar sit-
uation was observed with the chimeras ALASF341A/AONS and
AONS/ALASF341A, in which the phenylalanine at position 341 of
murine erythroid ALAS was substituted with an alanine (Table 2).
When the ALAS sequence was maintained intact and a mutation
of the Schiff base linkage-lysine residue was introduced into AONS
(i.e., K236A), the generated chimeric proteins, AONSK236A/ALAS or
ALAS/AONSK236A, could rescue the growth of HU227 cells in a med-
ium without ALA but not of R872 cells in a non-biotin supple-
mented medium (Table 2). Taken together, these findings are
consistent with the active site arrangement for ALAS/AONS de-
picted in Fig. 4D, in which the active sites responsible for ALAS
activity are built with ALAS residues, while those with AONS activ-
ity are made of AONS residues.
Oligomeric state of the ALAS/AONS chimera
Although ALAS/AONS and AONS/ALAS could be overproduced as
active chimeric enzymes in E. coli DH5a cells (data not shown),
only ALAS/AONS could be purified. Poor solubility and low stability
were among the factors preventing the purification of AONS/ALAS.
The molecular mass of each subunit of the WT ALAS and WT
AONS homodimers is ꢁ56 and ꢁ40 kD, respectively. Thus a single
chimeric subunit, derived from the fusion of an ALAS and AONS
polypeptide, was expected to be ꢁ96 kD. This is in agreement with
the molecular mass estimated by SDS–PAGE (Fig. 5, inset). The
molecular mass of the native ALAS/AONS chimera was determined
to be ꢁ182 kD (Fig. 5), consistent of a ‘‘homodimer’’ of ꢁ96 kD sub-
units. Further, coupled enzyme assays confirmed that the 182-kD
protein exhibits both ALAS and AONS activities, indicating that
the ALAS/AONS chimera is bifunctional as a homodimer of two
ꢁ96 kD chimeric polypeptide subunits.
Fig. 6. Absorption and fluorescence spectra of ALAS, AONS and ALAS/AONS. (A) UV–
visible absorption spectra. The inset includes the region from 300 to 510 nm.
Fluorescence emission spectra upon excitation at (B) 420 nm and (C) 330 nm and
fluorescence excitation spectra upon emission at 510 nm (inset B) and 385 nm (inset
C). For absorption spectra, protein concentrations were adjusted to 15
and ALAS) or 7.5 M (ALAS/AONS) and for fluorescence spectra, protein concentra-
tions were adjusted to 4 M (AONS and ALAS) or 2 M (ALAS/AONS) in 20 mM
lM (AONS
l
l
l
Hepes, pH 7.5 containing 10% glycerol. For (A)–(C), AONS (- -), ALAS (—), and ALAS/
AONS (–ꢃ–).
ible spectra of both ALAS and AONS exhibited maxima at 330 and
420 nm, their fluorescence spectra were examined for distinctive
features among ALAS, AONS and ALAS/AONS. With excitation at
420 nm, the PLP cofactor of either ALAS or AONS exhibited a fluo-
rescence emission maximum at 510 nm for ALAS and AONS
(Fig. 6B), albeit the magnitude of the 510 nm fluorescence emission
Spectroscopic characterization of AONS and ALAS/AONS
At pH 7.5, absorbance maxima at ꢁ330 and ꢁ420 nm were
observed in the ALAS/AONS chimera (Fig. 6A). Because the UV–vis-

114
T.D. Turbeville et al. / Archives of Biochemistry and Biophysics 511 (2011) 107–117
Table 4
ALAS activity: summary of steady-state kinetic parameters for ALAS and ALAS/AONS chimera.
kcat (minꢀ1
)
KGmly (mM)
23
5.5 0.8
kcat/KmGly (minꢀ1 mMꢀ1
)
KSmCoA
(
l
M)
kcat/KmSCoA (minꢀ1 Mꢀ1
l )
ALASa
ALAS/AONS
10
1
1
0.43 0.02
1.1 0.2
2.3 0.1
1.5 0.2
4.3 0.2
4.1 0.7
6.2 0.8
a
Data from Ref. [28].
maximum was more than three times greater for the AONS cofac-
tor than that of ALAS. Similar to AONS and ALAS, the PLP cofactor of
the ALAS/AONS chimera exhibited a fluorescence emission maxi-
mum at 510 nm upon excitation at 420 nm, and the magnitude
of this fluorescence emission maximum fell between the values ob-
served for the PLP cofactor of ALAS and AONS (Fig. 6B). Upon exci-
tation at 330 nm, the fluorescence emission spectra of ALAS and
AONS exhibited maxima at 385 and 430 nm, respectively, while
the fluorescence emission spectrum of the ALAS/AONS chimera
displayed a broad emission band between 385 and 430 nm
(Fig. 6C).
Steady-state and transient kinetics of ALAS/AONS
To examine the ALAS and AONS activities of the ALAS/AONS chi-
mera, the steady-state kinetic parameters of the chimeric protein
associated with both activities were determined using substrates
for ALAS and AONS (Tables 4 and 5). Regarding the ALAS activity,
the kcat decreased almost 40%, the catalytic efficiency for glycine
increased ꢁ1.4-fold, and the catalytic efficiency for succinyl-CoA
remained virtually the same relative to ALAS (Table 4). With re-
spect to the AONS activity, while the value for kcat decreased
approximately 50%, the catalytic efficiencies towards alanine and
pimeloyl-CoA of the ALAS/AONS chimera were similar to those of
AONS (Table 5).
Fig. 7. Kinetics of a pre-steady-state burst of ALA in the ALAS/AONS reaction. ALAS/
AONS (50 M) preincubated with glycine (140 mM) was quickly reacted with
succinyl-CoA (150 M) at 20 °C. The concentrations shown in parentheses are final
concentrations after mixing. The reactions were quenched with perchloric acid
(0.14 M) at various aging times, and the ALA concentration was determined. The
inset illustrates the first 0.6 s of the reaction time course. The first 0.7 s of the time
course is expanded while in the inset the time course is extended to 3.0 s. The curve
l
l
represents the best fit to Eq. (1) with a burst amplitude of 4.7 0.4
lM and a burst
rate of 13.2 2.6 sꢀ1
.
Using chemical quenched-flow, pre-steady-state experiments of
the ALAS reaction were performed under multi-turnover condi-
tions, to examine the extent of ALA production by ALAS/AONS
and ascertain the reactivity of the ALAS active sites in the ALAS/
AONS chimera (Fig. 7). The time course associated with ALA forma-
tion was biphasic, described by a rapid, exponential phase (i.e.,
burst phase) followed by a slower linear phase (i.e., steady-state
phase). The first turnover occurred at a rate of 13.2 2.6 sꢀ1, while
subsequent turnovers took place at a rate of 0.015 sꢀ1. The ob-
served pre-steady state burst clearly indicated that the rate-limit-
ing step in the overall ALAS reaction occurs after the chemical step,
ALA formation, and corresponds to release of the product from the
active site. While the values for the burst and the steady-state rates
were approximately 70% and 50% of those previously determined
for the wild-type ALAS-catalyzed reaction [33] the burst amplitude
of 0.10/active site was similar to that formerly observed with wild-
type ALAS [33]. Significantly, the ꢁ50% decrease in the value of the
steady-state rate agrees with that inferred from the experiments
performed under steady-state conditions (above). Thus, ALA is
formed and accumulated similarly on the active sites of ALAS
and ALAS/AONS, and the diminished steady-state ALAS activity of
the ALAS/AONS chimera in relation to that of ALAS must arise from
a step occurring after the reaction chemistry, presumably a
strained protein conformational change associated with product
release.
Discussion
ALAS and other fold-type I PLP-dependent enzymes function as
homodimers with two active sites per dimer; each active site is
created at the interface between the two monomeric subunits
[1,2]. The crystal structure of the R. capsulatus ALAS holoenzyme
revealed that the enzyme symmetrically binds two PLP molecules,
one at each active site [8]. Previously, we demonstrated that link-
ing the two subunits of ALAS into a single polypeptide chain
yielded a more-active enzyme that functioned as a single-chain di-
mer [23]. Although linking the N-terminus of one subunit and the
C-terminus of the adjacent subunit without the introduction of a
polypeptide linker did not affect the global conformation, changes
in the environment of the PLP cofactor altered the predominant
tautomeric form of the internal aldimine, which contributed to
the greater activity of the single-chain dimer [23]. However, it
was not clear if these changes affected the enzymatic activity of
each active site to a similar extent. To determine whether the
Table 5
AONS activity: summary of steady-state kinetic parameters for AONS and ALAS/AONS chimera.
kcat (minꢀ1
)
KPmCoA
(l
M)
kcat/KmPCoA (minꢀ1 Mꢀ1
l )
KAmlanine (mM)
kcat/KmAlanine (min1 mMꢀ1
)
AONSa
ALAS/AONS
3.6 0.6
1.7 0.3
0.5 0.4
0.25 0.05
7.2 1.3
6.8 1.8
25
10
2
1
0.14 0.03
0.16 0.03
a
Data from Ref. [29].

T.D. Turbeville et al. / Archives of Biochemistry and Biophysics 511 (2011) 107–117
115
two active sites in ALAS/ALAS contribute equally to enzymatic
activity, we characterized variants in which one of the two active
sites had no measurable enzymatic activity due to a mutation of
the conserved K313 residue that binds to the cofactor.
show that the variant heterodimer retained approximately 50% of
the activity observed in the ALAS homodimer [18]; that is, the
WT active site was not significantly affected by mutations at the
adjacent site, and the two sites appeared to work independently
in the ALAS homodimer. Thus, linking the termini of the two ALAS
subunits made the two active sites to function asymmetrically by
likely altering the intermolecular dynamics such that movement
of the active site loop for product release was greatly enhanced
at one active site and impaired at the other.
Spectral characterization of ALAS/ALASK313A and ALASK313A/ALAS
revealed asymmetric cofactor environments in the two active sites,
which was reflected in the disproportionate kinetic behavior of the
two active sites. In contrast to ALAS/ALAS and ALASK313A/ALAS, in
which the catalytic rates increased 5-fold and 2-fold, respectively,
relative to the values for WT ALAS, the catalytic rate decreased
approximately 50% for ALAS/ALASK313A. The concentration of cata-
lytically active sites in ALAS/ALASK313A and ALASK313A/ALAS was half
that of the ALAS/ALAS but, unlike the variations observed in the stea-
dy-state rates, the chemical rates were similar in all three forms. As
with ALAS/ALAS and WT ALAS [23,33], the pre-steady-state burst
kinetics results for the two K313A variants were consistent with
the rate-limiting step occurring after the reaction chemistry.
In ALAS, the rate-limiting step has been ascribed to the opening
of an active site loop that allows ALA release; the rate of this as-
signed loop opening closely corresponds to the steady-state rate
[33,38,41]. Mutations introduced in the 18-amino acid active site
loop yielded variants of ALAS with significantly increased rates of
loop opening and product release [41]. Thus, the variations ob-
served in the steady-state kinetic parameters of the single chain di-
mer are likely to be due to alterations in the energy barrier for the
conformational change required for ALA release. The ALAS crystal
structure indicates that the N-terminus of one subunit and the C-
terminus of the adjacent subunit are located near the surface on
opposite faces of the holoenzyme [8]. The strain resulting from
linking the remote N- and C-termini of two ALAS subunits in
ALAS/ALAS appears to increase the energy barrier for product re-
lease at one site while decreasing the barrier at the other; that is,
the steady-state enzymatic activity is enhanced at one active site
and hindered at the other. Consequently, the active sites contribute
asymmetrically to enzyme function.
Because the single-chain ALAS dimer showed structural plastic-
ity and had increased activity, we wondered whether the structural
plasticity would extend to single-chain chimeras constructed from
two members of the a-oxoamine synthase family, ALAS and AONS.
We also hypothesized that it might be possible to generate an en-
zyme with novel activity by creating hybrid ALAS/AONS active
sites. In vivo assays indicated that the ALAS/AONS and AONS/ALAS
chimeras possessed both ALAS and AONS activities. Thus, the chi-
meric protein had sufficient structural plasticity to achieve the
conformations necessary to produce both enzymatic activities.
The linking of two ALAS subunits did not significantly affect the di-
meric interface or the folding of the core in the individual subunits,
and the dissociation of the dimeric interface of ALAS and the ALAS/
ALAS single-chain dimer exhibited similar free energies and re-
sulted in stable intermediates that retained a substantial amount
of their secondary and tertiary structure [21,22]. Similarly, we
would not expect the linking of ALAS and AONS to strongly affect
the folding core or subunit interface of the two domains, and it is
not surprising that the two domains retained their overall struc-
tural character given that the entire ALAS and AONS polypeptides
were used in the creation of the chimeras.
Despite our initial hypothesis that the chimeric protein would
create chimeric active sites with potentially novel enzymatic activ-
ities, both ALAS/AONS and AONS/ALAS appeared to function as chi-
meric homodimers with functionally independent ALAS and AONS
modules. In these modules, two active sites exhibiting ALAS activ-
ity were built exclusively with ALAS residues, and two active sites
with AONS activity were built with AONS residues (Fig. 4D). The
chimeric proteins seem to assemble forming wild-type active sites.
The implication is that other interactions besides those at the ac-
tive site contribute to the recognition for the dimer interface, and
those recognition elements are specific for a particular enzyme.
Nonetheless, the dimerization of two chimeric polypeptides into
a bifunctional homodimer with functionally independent ALAS
and AONS modules suggests that the structural plasticity observed
Generally, the active sites in fold type I PLP-dependent enzymes
are equivalent and independent [2], although some examples of ki-
netic asymmetry have been documented. In glutamate-1-semial-
dehyde aminomutase (GSAM), allosteric interactions between
active sites lead to inactivation of the site in one subunit by the
activation of the site in the other subunit [42]. In aspartate amino-
transferase (AAT), dissimilar lattice contact in the crystalline en-
zyme contributes to kinetic asymmetry in the active sites,
although the active sites display kinetic equivalence in solution
[43]. In addition, heterodimeric variants created for a number of
complementation studies have contained asymmetric active sites
[18,44–46], but the creation of ALAS/ALAS is the first example of
engineered active site asymmetry without the introduction of ac-
tive site mutations in fold-type I PLP-dependent enzymes. This
linked dimer made it possible to alter and examine one active site
in the presence of the other, which is a difficult undertaking with a
‘‘two-polypeptide chain dimer’’.
We reasoned that the steady-state rates in the functioning
active sites of either ALAS/ALASK313A or ALASK313A/ALAS would be
50% of that of the ALAS/ALAS single chain dimer, in which both
sites contribute to enzyme function. Thus, the sum of the steady-
state rates of ALAS/ALASK313A and ALASK313A/ALAS would equal
that of ALAS/ALAS. Clearly, this was not the case. The unequal stea-
dy-state rates of ALAS/ALASK313A and ALASK313A/ALAS (ꢁ8% and
ꢁ40% of the steady-state rate of ALAS/ALAS, respectively) and the
sum of their rates at about 50% of the steady-state rate of ALAS/
ALAS led us to conclude that the two active sites do not contribute
equally to the steady-state activity of the non-mutated single-
chain dimer. Conversely, studies in the unlinked ALASK313A and
ALASR149A heterodimer, containing one non-active site with both
K313A and R149A mutations and one WT catalytically active site,
in ALAS can be extended to other members of the a-oxoamine syn-
thase family. It is possible that creating a chimera from individual
domains of each protein (for example, the N-terminal domain of
ALAS and the catalytic and C-terminal domains of AONS) would
produce a functional hybrid, but this remains to be tested.
Although we succeeded in expressing both the ALAS/AONS and
AONS/ALAS chimeras as active enzymes, we were only able to pur-
ify the ALAS/AONS chimera. The molecular mass of the native
ALAS/AONS chimera was consistent with that of a homodimer con-
taining two chimeric polypeptide subunits. The fluorescence spec-
tra exhibited by the ALAS/AONS chimera were distinct from those
of ALAS and AONS and were consistent with an enzyme exhibiting
a mixture of ALAS and AONS spectroscopic characteristics. The
ALAS and AONS steady-state kinetic activities were diminished
by roughly one-half in the chimera, and the catalytic efficiencies
were not impaired. The pre-steady-state kinetic analysis for the
ALAS reaction demonstrated that ALA formation in the ALAS sites
of ALAS/AONS was similar to that in ALAS, with the rate-limiting
step occurring after chemical catalysis.
The linking of two proteins or functional domains in natural and
de novo fusion proteins generally involves a peptide linker [47–49].
A primary goal of linker engineering is to effectively separate the

116
T.D. Turbeville et al. / Archives of Biochemistry and Biophysics 511 (2011) 107–117
two functional domains to avoid intermolecular strain and prevent
unwanted interactions between the two modules [47,48,50–52].
Because our objective was to facilitate interaction between the
subunits of ALAS/ALAS and the AONS and ALAS chimeras, only
two amino acids (Glu-Leu), which were introduced with the con-
struction of a restriction site between the cDNAs, link the two sub-
units. Like both ALAS single-chain dimeric variants, the linking of
the ALAS and AONS subunits appeared to change the energy barrier
associated with the structural rearrangement that occurs upon ALA
formation to allow product release. It is likely that the use of the
short dipeptide to link the ALAS C-terminus with the N-terminus
of either ALAS or AONS introduced intermolecular strain which al-
tered conformational flexibility.
Remarkably, the strain introduced with the short linker did not
significantly impair the catalytic efficiencies in any of the fusion
proteins. In fact, the catalytic efficiency for succinyl-CoA increased
roughly 15- and 30-fold in ALASK313A/ALAS and ALAS/ALAS, respec-
tively (Table 3). The short linker also appears to have altered the
intermolecular dynamics and caused different extent of the active
site loop motion at the two sites. While the loop motion and prod-
uct release were greatly facilitated at one site, they were hindered
at the other. However, since the degree of enhancement at one site
was greater than the degree of hindrance at the other site, the
resulting linked chain dimer (ALAS/ALAS) possessed greater en-
zyme activity than the ‘‘natural’’ wild-type homodimer (ALAS).
Generally, efforts are made to avoid the introduction of strain
when engineering hybrid proteins; however, with the increased
use of high-throughput protein engineering, the introduction of
intermolecular strain through linker domains may be a reasonable
approach to creating new proteins with enhanced or novel func-
tions. This might be particularly important if one takes into ac-
count that recruitment of domains and subunits, with alteration
of domain architecture and oligomeric state, seems to represent
one mechanism of how proteins evolve to acquire distinct func-
tions with a limited number of scaffolds [43]. Oligomerization
and domain fusion appeared to be a strategy adopted by transfer-
ases to shield hydrophilic ligands, contrasting to their homologous,
and perhaps parent, hydrolases, in which the ligand molecules are
exposed to water [43].
and Dr. Charlotte S. Russell (City University of New York) for the
E. coli HU227 strain. We also thank Ms. Michelle Grigsby for her
technical assistance in the construction of some of the expression
plasmids and purification of some batches of ALAS/ALAS.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.abb.2011.05.002.
References
[1] P. Christen, P.K. Mehta, Chem. Rec. 1 (2001) 436–447.
[2] A.C. Eliot, J.F. Kirsch, Annu. Rev. Biochem. 73 (2004) 383–415.
[3] G.C. Ferreira, J. Gong, J. Bioenerg. Biomembr. 27 (1995) 151–159.
[4] G.A. Hunter, G.C. Ferreira, Biochim. Biophys. Acta (2011) [Epub ahead of print]
PMID: 21215825.
[5] G. Layer, J. Reichelt, D. Jahn, D.W. Heinz, Protein Sci. 19 (2010) 1137–1161.
[6] G. Schneider, H. Kack, Y. Lindqvist, Structure 8 (2000) R1–R6.
[7] B.A. Yard, L.G. Carter, K.A. Johnson, I.M. Overton, M. Dorward, H. Liu, S.A.
McMahon, M. Oke, D. Puech, G.J. Barton, J.H. Naismith, D.J. Campopiano, J. Mol.
Biol. 370 (2007) 870–886.
[8] I. Astner, J.O. Schulze, J. van den Heuvel, D. Jahn, W.D. Schubert, D.W. Heinz,
EMBO J. 24 (2005) 3166–3177.
[9] A. Schmidt, J. Sivaraman, Y. Li, R. Larocque, J.A. Barbosa, C. Smith, A. Matte, J.D.
Schrag, M. Cygler, Biochemistry 40 (2001) 5151–5160.
[10] D. Alexeev, M. Alexeeva, R.L. Baxter, D.J. Campopiano, S.P. Webster, L. Sawyer, J.
Mol. Biol. 284 (1998) 401–419.
[11] M.C.C. Raman, K.A. Johnson, B.A. Yard, J. Lowther, L.G. Carter, J.H. Naismith, D.J.
Campopiano, J. Biol. Chem. 284 (2009) 17328–17339.
[12] H. Ikushiro, S. Fujii, Y. Shiraiwa, H. Hayashi, J. Biol. Chem. 283 (2008) 7542–
7553.
[13] H. Ikushiro, M.M. Islam, A. Okamoto, J. Hoseki, T. Murakawa, S. Fujii, I.
Miyahara, H. Hayashi, J. Biochem. (Tokyo) 146 (2009) 549–562.
[14] N. Jahan, J.A. Potter, M.A. Sheikh, C.H. Botting, S.L. Shirran, N.J. Westwood, G.L.
Taylor, J. Mol. Biol. 392 (2009) 763–773.
[15] T. Spirig, A. Tiaden, P. Kiefer, C. Buchrieser, J.A. Vorholt, H. Hilbi, J. Biol. Chem.
283 (2008) 18113–18123.
[16] A. Tiaden, T. Spirig, P. Carranza, H. Bruggemann, K. Riedel, L. Eberl, C.
Buchrieser, H. Hilbi, J. Bacteriol. 190 (2008) 7532–7547.
[17] A. Tiaden, T. Spirig, H. Hilbi, Trends Microbiol. 18 (2010) 288–297.
[18] D. Tan, G.C. Ferreira, Biochemistry 35 (1996) 8934–8941.
[19] O. Ploux, A. Marquet, Biochem. J. 283 (Pt 2) (1992) 327–331.
[20] G.C. Ferreira, P.J. Neame, H.A. Dailey, Protein Sci. 2 (1993) 1959–1965.
[21] A.V. Cheltsov, M.J. Barber, G.C. Ferreira, J. Biol. Chem. 276 (2001) 19141–
19149.
[22] A.V. Cheltsov, W.C. Guida, G.C. Ferreira, J. Biol. Chem. 278 (2003) 27945–
27955.
[23] J. Zhang, A.V. Cheltsov, G.C. Ferreira, Protein Sci. 14 (2005) 1190–1200.
[24] G. Han, K. Gable, L. Yan, M.J. Allen, W.H. Wilson, P. Moitra, J.M. Harmon, T.M.
Dunn, J. Biol. Chem. 281 (2006) 39935–39942.
[25] G. Han, S.D. Gupta, K. Gable, S. Niranjanakumari, P. Moitra, F. Eichler, R.H.
Brown, J.M. Harmon, T.M. Dunn, Proc. Natl. Acad. Sci. USA 106 (2009) 8186–
8191.
[26] G.C. Ferreira, H.A. Dailey, J. Biol. Chem. 268 (1993) 584–590.
[27] G.C. Ferreira, U. Vajapey, O. Hafez, G.A. Hunter, M.J. Barber, Protein Sci. 4
(1995) 1001–1006.
[28] J. Gong, G.A. Hunter, G.C. Ferreira, Biochemistry 37 (1998) 3509–3517.
[29] S.P. Webster, D. Alexeev, D.J. Campopiano, R.M. Watt, M. Alexeeva, L. Sawyer,
R.L. Baxter, Biochemistry 39 (2000) 516–528.
[30] P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H. Gartner, M.D.
Provenzano, E.K. Fujimoto, N.M. Goeke, B.J. Olson, D.C. Klenk, Anal. Biochem.
150 (1985) 76–85.
In summary, we constructed a dimeric enzyme from a single
polypeptide chain such that each active site could be studied inde-
pendently. We have shown that the two active sites in the ALAS/
ALAS single-chain dimer make asymmetrical steady-state contri-
butions to the activity of the enzyme. This and the reduced stea-
dy-state activity of the single-chain chimeras of ALAS and AONS
relative to the parent enzymes are likely to be caused by differ-
ences in conformational changes at product release, which in turn
are due to the strain introduced by joining the two subunits with-
out a linker region. Although the chimeric ALAS/AONS and AONS/
ALAS proteins did not form hybrid active sites, they were able to
form dimers with separate regions of ALAS and AONS activity.
Thus, the extensive structural plasticity seen in ALAS extends to
[31] G.A. Hunter, G.C. Ferreira, Anal. Biochem. 226 (1995) 221–224.
[32] S.P. Webster, D.J. Campopiano, D. Alexeev, M. Alexeeva, R.M. Watt, L. Sawyer,
R.L. Baxter, Biochem. Soc. Trans. 26 (1998) S268.
[33] J. Zhang, G.C. Ferreira, J. Biol. Chem. 277 (2002) 44660–44669.
[34] K.A. Johnson, Enzymes XX (1992) 1–61.
another member of the a-oxoamine family, AONS, and other inter-
actions in addition to those at the active site contribute to the rec-
ognition for the dimer interface of a specific enzyme.
[35] J.M. Li, O. Brathwaite, S.D. Cosloy, C.S. Russell, J. Bacteriol. 171 (1989) 2547–
2552.
Funding
[36] J. Gong, G.C. Ferreira, Biochemistry 34 (1995) 1678–1685.
[37] G.A. Hunter, G.C. Ferreira, Biochemistry 38 (1999) 12526–12531.
[38] G.A. Hunter, J. Zhang, G.C. Ferreira, J. Biol. Chem. 282 (2007) 23025–23035.
[39] A. Sasarman, M. Surdeanu, T. Horodniceanu, J. Bacteriol. 96 (1968) 1882–1884.
[40] A. Del Campillo-Campbell, G. Kayajanian, A. Campbell, S. Adhya, J. Bacteriol. 94
(1967) 2065–2066.
This work was supported by the National Institutes of Health,
USA (Grants # DK63191 and # GM080270 to G.C.F.).
[41] T. Lendrihas, G.A. Hunter, G.C. Ferreira, J. Biol. Chem. 285 (2010) 13704–13711.
[42] J. Stetefeld, M. Jenny, P. Burkhard, Proc. Natl. Acad. Sci. USA 103 (2006) 13688–
13693.
Acknowledgments
[43] H. Kirsten, H. Gehring, P. Christen, Proc. Natl. Acad. Sci. USA 80 (1983) 1807–
1810.
We thank Dr. Dominic Campopiano (University of Edinburgh)
for providing the Escherichia coli R872 strain and pET6HAONS
[44] J.J. Onuffer, J.F. Kirsch, Protein Eng. 7 (1994) 413–424.

T.D. Turbeville et al. / Archives of Biochemistry and Biophysics 511 (2011) 107–117
117
[45] D. Tan, T. Harrison, G.A. Hunter, G.C. Ferreira, Biochemistry 37 (1998) 1478–
1484.
[46] A.S. Tarun, A. Theologis, J. Biol. Chem. 273 (1998) 12509–12514.
[47] W. Wriggers, S. Chakravarty, P.A. Jennings, Biopolymers 80 (2005) 736–746.
[48] R. Arai, H. Ueda, A. Kitayama, N. Kamiya, T. Nagamune, Protein Eng. 14 (2001)
529–532.
[49] R.S. Gokhale, C. Khosla, Curr. Opin. Chem. Biol. 4 (2000) 22–27.
[50] H. Carlsson, S. Ljung, L. Bulow, Biochim. Biophys. Acta 1293 (1996) 154–160.
[51] H.S. Seo, Y.J. Koo, J.Y. Lim, J.T. Song, C.H. Kim, J.K. Kim, J.S. Lee, Y.D. Choi, Appl.
Environ. Microbiol. 66 (2000) 2484–2490.
[52] R. Arai, W. Wriggers, Y. Nishikawa, T. Nagamune, T. Fujisawa, Proteins 57
(2004) 829–838.