Biochemical and structural characterization of alanine racemase from Bacillus anthracis (Ames)
BMC Structural Biology
Biochemical and structural characterization of alanine racemase from Bacillus anthracis (Ames)
Rafael M Couago 2
Milya Davlieva 1
Ulrich Strych 0
Ryan E Hill 2
Kurt L Krause 2
0 Department of Biology and Biochemistry, University of Houston , Houston, TX , USA
1 Department of Biochemistry Rice University , Houston, TX , USA
2 Department of Biochemistry, University of Otago , Dunedin , New Zealand
Background: Bacillus anthracis is the causative agent of anthrax and a potential bioterrorism threat. Here we report the biochemical and structural characterization of B. anthracis (Ames) alanine racemase (AlrBax), an essential enzyme in prokaryotes and a target for antimicrobial drug development. We also compare the native AlrBax structure to a recently reported structure of the same enzyme obtained through reductive lysine methylation. Results: B. anthracis has two open reading frames encoding for putative alanine racemases. We show that only one, dal1, is able to complement a D-alanine auxotrophic strain of E. coli. Purified Dal1, which we term AlrBax, is shown to be a dimer in solution by dynamic light scattering and has a Vmax for racemization (L- to D-alanine) of 101 U/mg. The crystal structure of unmodified AlrBax is reported here to 1.95 resolution. Despite the overall similarity of the fold to other alanine racemases, AlrBax makes use of a chloride ion to position key active site residues for catalysis, a feature not yet observed for this enzyme in other species. Crystal contacts are more extensive in the methylated structure compared to the unmethylated structure. Conclusion: The chloride ion in AlrBax is functioning effectively as a carbamylated lysine making it an integral and unique part of this structure. Despite differences in space group and crystal form, the two AlrBax structures are very similar, supporting the case that reductive methylation is a valid rescue strategy for proteins recalcitrant to crystallization, and does not, in this case, result in artifacts in the tertiary structure.
Bacillus anthracis is a soil-dwelling, spore-forming,
Grampositive bacterium that is the causative agent of the
zoonotic disease anthrax. Although the disease is most
common in wild and domestic mammals, it can also
occur in humans when exposed to infected animals or
living spores . The severity of anthrax in humans depends
on the route of infection. Inhalation of B. anthracis spores
can lead to the most severe form of the disease,
historically associated with a case-fatality rate as high as 85%
[2,3]. The high mortality rate, the existence of a respiratory
route of infection and the great resistance of its spores has
made B. anthracis the subject of biological warfare
research programs in many countries for over 60 years .
The United States Centers for Disease Control and
Prevention (CDC) has classified anthrax as a category A
bioterrorism agent, posing the greatest possible threat to public
health and with the ability to spread across large areas .
In 2001, the Ames strain of B. anthracis was used in a series
of bioterrorist attacks that resulted in five fatalities and
cost billions of dollars to the US economy [6,7]. As B.
anthracis spores are resilient, remaining viable and
infective for many years, efforts to decontaminate affected
facilities are time-consuming and costly. Therefore, it
would be of significant importance to public health and
security to develop new strategies aimed at containing B.
anthracis spores upon their release into the environment.
Alanine racemase (EC 126.96.36.199) is an essential enzyme in
prokaryotes. The enzyme utilizes a pyridoxal
5'-phosphate (PLP) cofactor to catalyze the racemization of
Lalanine to D-alanine, an essential component of the
peptidoglycan layer in bacterial cell walls. The lack of alanine
racemase function in eukaryotes has made this enzyme an
attractive target for antimicrobial drug development [8,9].
In B. anthracis, the gene coding for alanine racemase, dal1,
is one of only four genes up-regulated during sporulation
. The dal1 gene product (AlrBax) is found on the
spores' outermost layer  and addition of alanine
racemase inhibitors has been shown to promote germination
of B. anthracis' spores  while endogenous production
of D-alanine, mediated by alanine racemase, inhibits
germination . Triggering the premature germination of B.
anthracis spores by spraying alanine racemase inhibitors
on affected areas may therefore be a strategy to speed
decontamination efforts and reduce the risk of infection
in humans. Further, AlrBax has recently been reported as an
immunodominant protein in a proteomic analysis of the
B. anthracis spore induced immunome [13,14]. Given the
importance of three-dimensional information in
structure-aided inhibitor design [15-17] and its growing role in
vaccine development [18-20], structural studies on AlrBax
are crucial to both of these goals.
The first structural studies of alanine racemases, which
were performed on the enzyme isolated from Geobacillus
(then Bacillus)stearothermophilus, revealed a homodimeric
enzyme with each monomer consisting of an / barrel
domain at the N-terminus and a C-terminal domain
composed mainly of strands. The active site is located at the
interface of the / barrel and domain near a PLP
cofactor forming an internal aldimine linkage to a lysine
residue [21,22]. Structural studies performed in the presence
of substrate analogs have identified the residues involved
in catalysis and shed light onto the enzyme's catalytic
mechanism . Moreover, structures of G.
stearothermophilus alanine racemase with the covalent inhibitors
alanine phosphonate and D-cycloserine (DCS) have
shown that enzyme inhibition is due to the covalent link
of these compounds to the PLP cofactor and helped
explain their non-specific inhibition of eukaryotic
PLPcontaining enzymes [24,25].
Alanine racemase structures from the human pathogens
Pseudomonas aeruginosa and Mycobacterium tuberculosis
have been solved  and both revealed further insights
into the enzyme's catalytic site that may lead to
identification of new, more specific inhibitors. The high-resolution
(1.45 ) structure of alanine racemase (DadX) from P.
aeruginosa showed evidence for an external aldimine linkage
of an unanticipated guest substrate in the active site ,
while the M. tuberculosis structure revealed that the narrow
entryway to the enzyme's active site is composed of highly
conserved residues distributed in layers beginning at the
PLP site . The structure of the DCS-producing
Streptomyces lavendulae has also been determined . S.
lavendulae can grow in the presence of DCS, and the structural
basis for the slower inhibition rate of DCS on S. lavendulae
Alr has been attributed to the enzyme's larger and more
rigid active site .
Here we report the cloning and characterization of the two
genes, dal1 and dal2, from the B. anthracis genome with
sequence similarities to other bacterial alanine racemase
genes. Although expression of dal2 in a heterologous
system failed, we have successfully expressed and purified the
gene product of dal1, which we term AlrBax, and performed
its kinetic and structural characterization.
Recently another group has reported that B. anthracis
alanine racemase crystallization required reductive
methylation . Interestingly we have not found this to be the
case. However, the availability of both structures, one
with and one without methylation, allows for a careful
comparison to be performed. Reductive methylation has
been employed previously to obtain atomic structures for
proteins recalcitrant to crystallization [29-33]. Due to its
reported successes, this method is becoming more utilized
[28,34,35]. Nevertheless, there may be concerns as to how
methylation impacts protein structure. Our analyses of
both structures suggest that despite differences in space
group and crystal lattice, reductive methylation does not
significantly alter the structure of alanine racemase from
Results and Discussion
Sequence analysis of the Bacillus Dal proteins
The sequences for both dal1 and dal2 genes amplified in
our laboratory from B. anthracis (Ames) genomic DNA are
100% identical to those previously deposited in
GeneBank (dal1 GeneID: 1087014 and dal2 GeneID:
30262102) . The protein sequences encoded by dal1
and dal2 both contain the characteristic motifs expected
for members of the alanine racemase family, such as a
PLP-binding site near the N-terminus, the two key
conserved catalytic amino acid residues Lys41 (AlrBax
numbering) and Tyr270, and a set of conserved residues making
up the entrance corridor to the alanine racemase active
site  (Figure 1). The gene product of dal1, which we
term AlrBax, is identical to the alanine racemase protein
previously associated with germination in B. anthracis
spores  and shares 57% amino acid identity with
AlrGst. Dal2, on the other hand, shows 41% sequence
identity to AlrBax and 40% identity to AlrGst.
In order to confirm that both dal1 and dal2 genes encode
functional alanine racemases we expressed these genes in
a D-alanine auxotrophic strain of E. coli, MB2795 .
Expression of the dal1 gene from B. anthracis or dadX from
P. aeruginosa fully restored the wild-type phenotype. Cells
transformed with pET28-TEV failed to grow, as did those
transformed with the B. anthracis dal2 gene (data not
Overexpression, purification and biochemical
characterization of Dal proteins
We used strain BL21(DE3), pLysS of E. coli to express dal1
and dal2 recombinant gene products. While dal1 was
expressed successfully, the expression of dal2 failed
repeatedly, even when conditions such as temperature,
IPTG concentration, or strain background were changed
(data not shown). Sequencing of the plasmid construct
revealed no obvious errors, and our expression system has
been successfully used for numerous proteins in the past.
We have no conclusive explanation for our inability to
express dal2 at measurable levels in E. coli. While the orf
appears to encode an alanine racemase enzyme, it clearly
is not expressed in the T7 overexpression system, possibly
also explaining the lack of complementation observed in
(SFAtirgluruMcrtteubr)1e,-Sb.alsaevdenadluiglnaem(eAnltrSolfa)alandinPe. raaecreumginaosesasfr(DomadBX.Paanoth)racis (AlrBax), G. stearothermophilus (AlrGst), M. tuberculosis
Structure-based alignment of alanine racemasesfrom B. anthracis (AlrBax), G. stearothermophilus (AlrGst), M.
tuberculosis (AlrMtb), S. lavendulae (AlrSla) and P. aeruginosa (DadXPao). The initial alignment was performed using
EXPRESSO (3DCoffee)  and adjusted manually upon inspection of the superimposed structures. An asterisk marks the
location of the Lys residue bound to PLP, the diamond marks the location of the Tyr residue that functions as the second base
in the racemase reaction, a bullet denotes the location of the carbamylated lysine found in other alanine racemase structures
and replaced by a chloride ion on AlrBax. I and M denote residues found in the middle or the inner layer of the active site
entryway along with their position in the entryway.
our earlier experiments. Given that, to our knowledge,
there are no reports characterizing dal2 in the literature,
we are led to believe that this gene is not usually expressed
in its homologous host. As overproduction of Dal2Bax
failed, all subsequent work was performed with dal1 to
yield a product, which we term AlrBax. AlrBax was purified
to homogeneity and displayed a single peak on molecular
Previous studies have suggested AlrBax might exist partly as
a tetramer in solution . We have used dynamic light
scattering (DLS) to determine that AlrBax has a
hydrodynamic radius of 3.7 nm, corresponding to a molecular
weight of 93 kDa. As the calculated molecular weight of
AlrBax is 43.7 kDa, this enzyme is unambiguously a dimer
(ca. molecular weight 87.4 kDa) in solution under the
conditions of this experiment. These measurements were
made on a monodisperse solution of AlrBax in which
99.9% of the mass was accounted for by the single peak at
We find that purified AlrBax has a Km for D-alanine of 2.8
mM and a Vmax of 101 U mg-1, where one unit was defined
as the amount of enzyme that catalyzed racemization of 1
mol of substrate per minute. These kinetic parameters
for racemization of L- to D-alanine of AlrBax fall in the
range of what has been observed before for other bacterial
alanine racemases [38-40]. Interestingly, despite the high
identity levels observed for residues in the active site of
AlrBax and AlrGst (Figure 1), the Vmax for the anthrax enzyme
is one order of magnitude lower than the one reported for
the G. stearothermophilus enzyme and closer to that
observed for alanine racemases isolated from other
pathogenic organisms. Our kinetic characterization reinforces
previous observations that there is a very wide dynamic
range in kinetic constants for alanine racemase, despite
the sequence and structural similarities of their active
Description of the Overall Structure of AlrBax from B.
Consistent with other alanine racemases, the tertiary
structure of AlrBax is a homodimer formed by
head-to-tailassociation of two monomers (Figure 2). Each monomer
is crystallographically distinct in this crystal form (Table
1), but the two monomers have very similar folds. The
rms difference obtained for their C atoms after
leastsquares superposition is 0.22 . AlrBax monomers consist
of two structurally distinct domains. Residues in the
N-terminus (16245) fold into an eight-stranded / barrel,
while the C-terminal residues (246389) and the first 15
N-terminal amino acids are part of a predominantly
structure. The homodimer displays two active sites,
formed by residues from the N-terminus of one monomer
and residues from the C-terminus of the other monomer.
The PLP cofactor forms a covalent bond to Lys41 and
points at the center of the / barrel. As previously
observed for AlrGst , extra density was present in the
active of AlrBax, which we model here as a molecule of
Structural comparisons of AlrBax with closely related
Below we compare AlrBax to the highly active Alr from the
non-pathogenic bacterium G. stearothermophilus (AlrGst) as
well as the less active Alrs from pathogenic bacteria P.
aeruginosa (DadXPao) and M. tuberculosis (AlrMtb). We also
compare AlrBax to the Alr from the DCS-producing
bacteria S. lavendulae (AlrSla). These enzymes share between 26
and 57% sequence identity (Figure 1 and Table 2). The
crystal structure of native AlrBax reveals some structural
features that may be responsible for its slower catalytic
rate and suggests regions that might be targeted in
designing inhibitors of this enzyme.
As noted, the B. anthracis AlrBax secondary structure and
general fold closely resembles that seen for other alanine
racemases . However, there are a few small
topological differences between the structures of AlrGst and AlrBax.
AlrBax is five residues longer than AlrGst; three of the five
extra residues in AlrBax extend Helix 8 by one turn; while
the remaining two extra residues locate to the very
N-terminus of AlrBax. Helix 8 does not take part in the enzyme's
active site nor does it make intermonomer contacts,
therefore, we do not anticipate this secondary structure to play
a critical role in AlrBax function.
Least-squares superposition of C atoms from N- and
Cterminal domains from AlrGst, AlrSla, AlrMtb and DadXPao to
the equivalent domains in AlrBax reveals average rms
differences ranging from 1.10 to 2.30 . The rms differences
correlate with sequence identity levels (Table 2).
Superposition of the N-terminal domains of AlrBax and AlrGst
reveals significant C deviations ( 1.8 ) for residues in
three loops (residues 121125, between H6 and S6;
residues 198202, between H8 and S8; residues 215219,
between H9 and S9) and residues 148158 on H7. These
regions all locate to the protein surface and have no
reported role in homodimer formation or substrate
binding and catalysis. On the other hand, superposition of the
C-terminal domains of AlrBax and AlrGst shows no regions
with C rms differences greater than 1.4 .
AlrBax and AlrGst have a similar hinge angle between N- and
The overall rms differences among various bacterial
alanine racemases (Table 2) suggest that despite their
topological similarity there are notable structural differences
between their individual domains. It has been reported
previously that the hinge angle between N- and
C-terminal domains varies among different alanine racemases
. It is due to this difference that monomers from
fRFriobgmbuorBne. ra2enpthrreasceinstation of the dimer of the alanine racemase
Ribbon representation of the dimer of the alanine
racemase from B. anthracis. The PLP co-factor is shown
as a ball and stick model. Monomers are shown in different
colors. N and C indicate the position of the C- and N-termini
of one monomer; primed letters denote the termini for the
ferent alanine racemases cannot be optimally
superimposed onto each other as a whole. While a single hinge
angle comparison was sufficient for the pair-wise analysis
previously given, the inclusion of more racemase
structures has let us to consider a new system of hinge angle
description. In this system, following superposition of the
N-terminal domains, we define the shift in the C-terminal
domains of one racemase compared to another through
two rotation angles. One is measured relative to a plane
parallel and one relative to a plane perpendicular to the
PLP ring (Figure 3). The hinge rotations relative to the
Nterminal domain may be relevant for enzyme activity as it
could influence the position of the second catalytic
residue, Tyr270' (primed numbers denote residues found in
the second monomer). For the planes parallel and
perpendicular to the PLP ring the rotation for the C atom of
Tyr270' from AlrBax compared to the structurally
equivalent atom in AlrGst, AlrMtb, AlrSla and DadXPao is 0.4/2.7,
3.9/8.2, 8.7/2.3 and 10.3/5.9, respectively (Figure 3).
Given that AlrGst and AlrBax have the most similar hinge
angles we have compared these two structures with the
other Alrs in order to establish which regions are
responsible for the hinge angles in various Alrs.
Differences in hinge angles between various Alrs were first
reported by us for AlrGst and DadXPao and attributed to
specific polar interactions present only in AlrGst. These
interactions are mediated by polar side chains of residues
Asp68 and Arg89 of the first AlrGst monomer and the polar
side chain of Asn379 and main chain O from Phe4 and
main chain N from His5 of the second monomer .
Here in AlrBax, we find equivalent polar interactions
facilitated by structurally analogous residues; Asp70 and
Arg93 of the first AlrBax monomer and Phe6, Tyr7 and
Asn384 of the second monomer. An additional
interaction takes place between Asp77 from Helix 4 and Leu386
and Ile389 in AlrBax. Therefore, the polar contacts that
have been proposed to mediate the hinge angle in AlrGst
are also present in AlrBax, and as noted above these two
structures have the most similar hinge angles. On the
other hand, these polar contacts are not seen for AlrMtb,
AlrSla and DadXPao. Figure 4 shows that the side chains of
residues in AlrBax taking part in intra-monomer
interactions make extensive contacts with residues on the second
monomer, when compared with structurally equivalent
residues in AlrSla. AlrMtb, AlrSla and DadXPao are shorter
than AlrGst and lack structurally equivalent residues to the
C- and N-terminus of the longer enzymes (Figure 1). For
example, AlrSla has no analogous residue to Asn384 in
AlrBaxas the peptide chain is only 380 residues long.
Moreover, the structurally equivalent residue for the polar
arginine is Gly89. It is not surprising that the hinge angles
of these three deviate most from AlrGst.
This analysis notwithstanding, the relevance of the hinge
angle to enzyme catalysis in alanine racemase remains
unclear. Although the Vmax values reported for all alanine
racemases studied to date vary by over three orders of
magnitude, it is not straightforward to attribute these
differences to hinge angle. Differences in hinge angles
certainly alter the relative orientation of the two active sites
in the dimer, but affect very little the geometry of each
active site as indicated in Table 2. Further, the Vmax for
AlrGst and AlrBax enzymes varies by more than 10 fold,
despite having similar hinge angles. Altering the hinge
Numbers in parenthesis denote sequence identity with AlrBax. aCalculated using monomer A. bCalculated using residues 4245. cCalculated using
residues 246389. dCalculated using residues 3945, 6367, 8488, 103107, 127140, 163171, 203210, 221228, 356363 from monomer A
and 268271 and 314319 from monomer B.
FDiigffuereen3ces in hinge angle between various alanine racemases
Differences in hinge angle between various alanine
racemases. Structural alignment of the N-terminal domain
/ barrel from B. anthracis (green), G. stearothermophilus
(red), M. tuberculosis (pink), S. lavendulae (blue) and P.
aeruginosa (yellow) shows that the hinge angle between N- and
Cterminal domains can vary. N- and C-termini are labeled N
and C and colored according to their respective structures.
The PLP cofactor for AlrBax is shown as a ball and stick model
angle of this enzyme experimentally through mutation or
cassette swapping may resolve this issue.
Intermonomer contacts and surface area
The dimer interface in alanine racemase is an important
area for structural analysis. Only the dimeric form of the
enzyme is catalytically active . Therefore, interface
residues are critical in forming a functional active site.
Certainly the interface functions to correctly position the
second catalytic tyrosine residue from the opposite
monomer on top of the active site. In addition, both
monomers contribute to the overall composition of the alanine
entryway and binding pocket. Loss of interface contacts
would alter this arrangement and could be used as a
strategy to inhibit Alr activity. Disruption of dimer interfaces is
becoming more common and has been successfully used
recently for drug targets in HIV and HCV [42-44].
Despite large differences in hinge angles, the location of
interface residues in various Alrs is very similar (Figure 5).
In Figure 5, the C atoms for residues taking part in
intermonomer contacts in various Alrs are shown as colored
spheres. The positions for the various spheres were
obtained following two independent structural
alignments, one using only atoms from the N-terminal domain
and the other using only atoms from the C-terminal
domain, and then plotted onto a ribbon diagram of AlrBax.
If the position of residues taking part in intermonomer
contacts is conserved among various Alrs, we would
expect the colored spheres to form tight clusters,
containing superimposed red, green, blue, yellow and pink
spheres. Indeed, as shown in Figure 5, most of the
intermonomer contacts from various Alrs are found in clusters
and thus are conserved among various Alrs. It is important
to keep in mind that the number of residues taking part in
intermonomer contacts varies among the analyzed Alrs.
For AlrBax, 94 of its 389 residues take part in
intermonomer contacts and both N- and C-terminal domains
contribute an almost equal number (44 and 50, respectively)
of residues to the interface. The total number of residues
in the interface of AlrGst, AlrMtb, AlrSla and DadXPao is
slightly smaller than in AlrBax. Nevertheless, for all
analyzed structures, both domains contribute almost equally
to the monomer-monomer interface.
At its dimer interface, the AlrBax structure displays a larger
surface area and higher number of polar interactions than
AlrGst, AlrSla and DadXPao (Table 3). Not surprisingly, most
of the additional buried surface area observed for AlrBax
results from the interactions involving N- and C-terminal
residues described in the hinge angle analysis above. If
residues from the N-terminus (410) and C-terminus
(383389) of AlrBax are excluded from the calculation, the
intermonomer surface area of AlrBax is reduced from 3,500
to 2,500 2, making it similar to the values found for AlrSla
and DadXPao (~2700 2) (Table 3).
AlrBax PLP-binding and active site
As observed for other Alrs, the active site of AlrBax is
formed by residues from both monomers, with the two
catalytic bases Lys41 and Tyr270' found in different
monomers. In the AlrBax structure, Lys41 is seen covalently
linked to the PLP cofactor. As was observed for one of the
AlrGst structures (1sft) , we have identified extra
density in the active site of AlrBax, which we have modeled as
a molecule of acetate. Acetate, which was present in our
crystallization solution, is an inhibitor of Alr  and its
carboxylate group is thought to bind the enzyme active
site in the same way the carboxylate group from alanine is
expected to do . The oxygen atoms from the acetate
molecule in our model are within hydrogen bonding
distance to the side chain oxygen from Tyr289', the main
chain nitrogen from Met317' and, perhaps more
importantly, to the side chain nitrogen atom from the catalytic
Lys41 residue (Figure 6).
The identity and position of active site residues is strongly
conserved among various Alrs (Table 2). As a result, the
hydrogen bonding network found for the PLP molecule in
the active site of AlrBax is similar to the one observed for
other Alrs. In AlrBax, side chain atoms from Tyr45, Arg138,
Arg24, His168, Ser209 and Tyr359 establish hydrogen
bonds to atoms in the PLP cofactor (Figure 6). These
residFMiiaoggluerarcmeula4orfstuhrefaoceppforsitoenemmonoonmomerer of the alanine racemases from B. anthracis (A) and S. lavendulae (B) docked into the ribbon
Molecular surface for one monomer of the alanine racemases from B. anthracis (A) and S. lavendulae (B)
docked into the ribbon diagram of the opposite monomer. AlrBax has extended intermonomer contacts at its C- and
N-termini that are not present for other alanine racemases like the one from S. lavendulae. Position of residues taking part in
intermonomer contacts are shown in purple and yellow for AlrBax and green and red for AlrSla. For AlrBax, residues on Helices
4 and 5 and on N- and C- termini that make intermonomer polar contacts are shown as sticks. Equivalent residues taking part
in intermonomer contacts are also shown as sticks for AlrSla. Positions for the N- and C- termini, Helix 4 and Helix 5 are
indicated by arrows.
dues are strictly conserved for AlrGst, AlrMtb, AlrSla and
DadXPao and have similar orientations in the PLP-binding
site of their respective enzymes. The PLP in AlrBax also
hydrogen bonds with main chain atoms from Ser209,
Gly226 and Ile227. The first two of these residues is
strictly conserved in AlrGst, AlrMtb, AlrSla and DadXPao. The
third would be as well but in AlrSla, the Ile227 is replaced
by a leucine residue. Perhaps a more significant difference
is the presence in AlrSla and AlrMtb of a tryptophan residue
in place of AlrBax Leu87. A tryptophan residue at this
position is one of the differences found between the active
sites of the slower enzymes from M. tuberculosis and S.
lavendulae and the faster Alr from G. stearothermophilus. In
AlrSla and AlrMtb, the N atom of this tryptophan makes a
water-mediated hydrogen bond to O3 from PLP.
Although this extra interaction may have a role in catalysis
it does not seem to reduce the size of the AlrSla and AlrMtb
active sites as the loop that harbors this tryptophan
residue is shifted away (~2.1 ) from the PLP cofactor when
compared to the same loop in AlrBax. Mutagenesis studies
could thus be performed in order to evaluate the impact
of this tryptophan residue for enzyme catalysis.
One striking difference in the active site involves Asn131,
which in other alanine racemases is generally a
carbamylated lysine that participates in a hydrogen bond
with the residue homologous to Arg138. In AlrBax,
however, we note a prominent chloride ion that is located near
Arg138 in the active site (Figure 6). This chloride ion has
not been described in Alr structures from other species
and it was originally modeled by us as a water molecule.
However, the resulting low B-factor (~10 2) and its
hexacoordination with three water molecules and atoms N2
from Asn131 and N and N2 from Arg138 suggested the
presence of a chloride ion. Notably, there is no chloride
present in the crystallization buffer and we can only
assume that the enzyme binds so tightly to this halide that
it is carried over from the enzyme's purification. The
chloride ion is also observed on the AlrBax structure obtained
following lysine reductive methylation . The presence
of a chloride ion in two independent structures reinforces
Position of residues taking part in intermonomer
contacts is highly conserved among various Alrs,
despite differences in hinge angles. Following a
structural alignment of the N-terminal domains of various Alrs,
the position for the C atoms from residues that take part in
intermonomer contacts and are in the N-terminal domain
(shown as colored spheres) was plotted on the main chain
representation of AlrBax (shown in green). Likewise, the
position for the C atoms from residues that take part in
intermonomer contacts and are at the C-terminal domain (shown
as colored spheres) were plotted on the main chain
representation of AlrBax after a structural alignment of the
C-terminal domains of various Alrs. Residues are colored
according to the legend on figure 3. The PLP cofactor from
AlrBax is shown as a ball and stick model. N and C indicate the
position of the C- and N-termini in the monomer.
the idea that this ion plays an important structural role in
AlrBax. Other Alrs have a negative charge at the same
position, but the charge has always been from a carbamylated
lysine residue (Figure 6). In the AlrMtb structure a
carbamylated lysine was not noted but the side chain density
for this lysine was poor. Like the chloride ion in AlrBax, the
carbamyl group found in other Alrs hydrogen bonds with
N and N2 from the active site arginine (Arg138 in
AlrBax), thus positioning this residue in the active site. The
general conservation of the modified lysine residue
among various Alrs and its role in positioning the active
site arginine indicates that the presence of a negative
charge at this position is critical for enzyme catalysis. As
AlrBax lacks the conserved lysine residue necessary for
carbamylation it has apparently drafted a chloride ion to fill
the same role for this species. It is open to speculation
whether the addition of chloride chelators like SPQ
(6methoxy-N-(3-sulfopropyl)-quinolinium) would affect
the enzyme activity and whether it might be possible to
design specific inhibitors for AlrBax based on this unique
In AlrBax in addition to the interactions facilitated by the
chloride ion, Arg138 is further positioned by the side
chain oxygen of Thr316'. Further, an alignment of 105
Alrs, having between 24% to 99% sequence identity to
AlrBax, revealed that the presence of an asparagine at the
equivalent position to Asn131 in AlrBax is always
accompanied by the presence of a threonine residue equivalent
to Thr316' (data not shown) suggesting that this
interaction with Arg138 would be a conserved feature of alanine
racemases with active site structural chlorides. Sequences
of Alrs that contain a lysine in position 131 almost always
have an accompanying serine or a cysteine residue in
position equivalent to AlrBax Thr316'. In the case of AlrPao this
serine is involved in an equivalent active site arginine
interaction. The exception to this latter observation is
AlrSla which has an alanine at this position. It is important
to note that there is not really a specific chloride-binding
motif as the residues that interact with Cl- in AlrBax are the
same that interact with the carbamylated lysine in the
The active site entryway of AlrBax
Residues from loops in the / barrel domain of one
monomer and residues from the C-terminal domain of
the second monomer make up an entryway to the active
site and the PLP binding site. The active site entryway of
Alr has been previously divided in inner, middle and
outer layers, starting from the PLP binding pocket and
moving towards the protein surface . Residues in the
inner and middle layers show strong conservation among
various Alrs . For AlrBax, residues Tyr270', Tyr359,
Tyr289' and Ala172 constitute the inner layer, while
residues Arg314', Ile357, Arg295' and Asp173 make up the
middle layer. These residues are absolutely conserved
between AlrBax and AlrGst, AlrMtb, AlrSla and DadXPao. The
outer layer for the active site entryway of various Alrs
displays less conservation, but in this region AlrBax contains
Intermonomer surface area (2)
an Asn271' while AlrGst, AlrMtb, AlrSla and DadXPao contain
a glycine. As a result of this substitution, the entryway is
somewhat more restricted than the ones observed for
other alanine racemases. Finally, for AlrBax a conserved
pair of acidic residues (Asp-Glu) is found at positions 173
and 174, which are located in the middle and outer layers
of the entryway. Identical residues are found in the same
position for AlrGst, AlrSla and DadXPao, but for AlrMtb, a
much slower alanine racemase, these two residues are
(Asp-Lys). This site has recently been shown to be
important catalytically, as making this Asp-Glu to Asp-Lys
change at the same position in E. coli alanine racemase has
been shown to significantly decrease its catalytic rate .
Structural comparison of native and reductively
methylated alanine racemases from B. anthracis
Recently, the structure of AlrBax after reductive methylation
of its lysine residues (AlrBaxRM) has been reported . In
that report, the unmodified protein failed to crystallize.
Scientists at the Oxford Protein Production Facility
(OPPF) and the York Structural Biology laboratory
reported that extensive crystallization trials
(approximately 800 conditions) with native AlrBax proved
unsucFOirgguarneiza6tion of the active site residues in B. anthracis Alr is facilitated by a chloride ion
Organization of the active site residues in B. anthracis Alr is facilitated by a chloride ion. (A) Electron density map
(contoured at 1.5 in the final refined 2Fo-Fc map) showing details of the active site for AlrBax. (B) Structural alignment of
residues making the active site of various Alrs (TB structure was not included). For all available Alr structures, Arg138 makes polar
contacts to the PLP and, possibly, to the substrate. In AlrBax this arginine residue is positioned in the active site by a chloride ion
(Cl-). Polar contacts between the chloride ion and Asn-131 and Arg-138 are shown in Panel A by dashes. For all other alanine
racemase structures available to date the equivalent interactions are mediated by a carbamylated lysine (shown in Panel B).
Residues in the active site of various Alrs are shown as a stick model. In Panel A, the acetate molecule and the modified lysine
residue (LLP) are depicted as ball and stick models; carbons are colored in green, nitrogen in blue, oxygen in red, phosphate in
orange, sulfur in yellow and the chloride ion is depicted as a light green sphere. In Panel B residues are shown as stick model
and are colored according to the legend on figure 3; the PLP cofactors are shown as ball and stick models. In both panels,
primed numbers denote residues from the second monomer.
cessful and that reductive lysine methylation was essential
for crystallization of the protein [34,46]. Based on data
from mass spectroscopy and on the methylated crystal
structure of AlrBax, Au and colleagues concluded that the N
terminus and 18 out of the 20 lysines in AlrBax were
methylated after the protein was treated with
dimethyl-amineborane complex and formaldehyde.
Reductive methylation modifies all free primary amines in
a protein molecule (NH groups from lysine residues and
the N terminus) to tertiary amines. This modification of
lysine residues, especially those found on the protein
surface, offers an opportunity to change a protein's
crystallization properties and is a proven method to rescue
proteins recalcitrant to crystallization [28-33,35,47].
However, there are few structural studies showing that
reductive alkylation does not alter a protein's structure,
especially of proteins that do not readily crystallize. One
study  reporting on the effects of reductive lysine
methylation on HEW lysozyme found that crystals were
formed under different conditions and with a different
crystalline lattice than observed for the unmodified
enzyme. Nevertheless, the structures of both modified
and unmodified enzymes showed no significant
structural differences and their superimposed C atoms had an
rms difference of only 0.4 . The availability of native
and modified structures for AlrBax, therefore, offers
another opportunity to evaluate the impact of reductive
lysine methylation, this time on a protein more
recalcitrant to crystallization.
In our hands, AlrBax protein readily formed small crystals
using commercially available crystallization screens.
Notably our form contains eight additional residues at the
C-terminus that remain following cleavage of a
C-terminal His-tag using TEV protease. These residues are not
involved in crystal contacts, but still could have an
influence on crystallization. Our initial crystallization
conditions required extensive fine-tuning, and the addition of
the glutathione additive proved important for obtaining
diffraction quality crystals. Moreover, finding the proper
conditions for freezing AlrBax crystals without
compromising diffraction quality proved challenging. For simplicity's
sake we have referred to this form of AlrBax as
unmethylated or native. Our review of the expression and
purification protocols for both native and alkylated enzymes
suggests that they were very similar. Also, modified and
unmodified AlrBax crystallize under similar conditions,
despite a reported small reduction in the isoelectric point
and the expected changes in the surface properties of
AlrBax . Both proteins were crystallized in the presence of
PEG (18% PEG 8000 for the native and 25% PEG 3350 for
the modified protein), high salt concentrations (0.2 M
sodium acetate for the native and 0.2 M magnesium
chloride for the modified protein) and at the same pH, 6.5.
Interestingly, the modified enzyme was crystallized at 60
mg/ml while the native structure was obtained from
crystals grown at 15 mg/ml.
Despite similar crystallization conditions native and
modified AlrBax crystals show different crystalline lattices
and solvent content. Native AlrBax crystals are monoclinic
with space group P21 and unit cell parameters a = 49.6 ,
b = 141.3 , c = 60.1 and = 103.11. On the other
hand, crystals for the methylated enzyme are
orthorhombic in space group P212121 with cell dimensions of a =
57.6 b = 88.4 and c = 139.0 . Crystals for the
modified enzyme display a lower solvent content (38% vs.
48%) and a higher packing density (1.99 3/Da vs. 2.35
3/Da) than native crystals.
Crystal contacts comparison
The total surface area found in crystal contacts for the
reductively methylated enzyme is 1.7 times larger than
that found for the native enzyme (1529.7 2 vs. 918.4 2,
respectively). Further, these contacts are often mediated
by methylated lysine residues found at the protein surface
(Figure 7). In monomer A from AlrBaxRM, 6 out of the 18
modified lysines contact protein atoms from both
monomers in adjacent asymmetric units. For monomer B, 9
modified lysines engage in crystal contacts; contacting
oFDfiigfsfuecrreoenn7cdeariny carmysintaelscoonntAaclrtBsaxfollowing reductive methylation
Difference in crystal contacts following reductive
methylation of secondary amines on AlrBax. The
position of C atoms from residues making crystal contacts are
shown as colored spheres superimposed onto the ribbon
diagram of AlrBax (shown in green). Methylated lysines
involved in crystal contacts are shown in red; other residues
involved in crystal contacts for the methylated structure only
are shown in yellow. Residues implicated with crystal
contacts for the native structure only are shown in blue.
Residues found to make crystal contacts in both structures are
shown in green. N and C indicate the position of the C- and
N-termini of one monomer; primed letters denote the
termini for the second monomer.
protein atoms in both monomers from symmetry related
protein molecules. Interestingly, methylated lysine 202
from monomer A contacts the same residue from
monomer B in a symmetry related molecule. In Figure 7, the
location of the C atoms from residues taking part in
crystal contacts for both the native and methylated structure is
shown as colored spheres. Different colors were used for
various categories of contacts. Yellow and red spheres are
for contacts observed only in crystals of the methylated
protein, while blue spheres are found only in the crystals
of the native protein. Contacts found in both crystal forms
are shown as green spheres. Figure 7 illustrates that
crystals of the methylated AlrBax contain more residues taking
part in crystal contacts, and as noted above, modified
lysine residues, shown as red spheres, make many of these
crystal contacts. In this case of AlrBax reductive
methylation does change the protein surface in a way to promote
the formation of a more extensive and apparently more
ordered crystalline lattice than that found for the native
The surfaces of the modified and native AlrBax crystals are
also different in terms of metal and halide content. Four
magnesium and three chloride ions were found on the
surface of modified AlrBax and take part in crystal contacts.
For the native AlrBax structure we did not identify any
metal or halide ions at equivalent positions. Furthermore,
the temperature factors for these surface ions are quite
low, with four less than 20 2, and many are involved in
extensive electrostatic interactions. Perhaps the presence
of additional metal ions observed exclusively for the
methylated crystal form of AlrBax acts to compensate for
the loss of positive charges at the protein surface.
Most importantly, reductive methylation did not alter the
overall fold of AlrBax. Structural alignment of methylated
and native AlrBax shows no significant difference in their
overall structures. For the individual monomers the rms
difference between their C atoms is just under 0.4 .
Alignment of the active site residues from the two AlrBax
structures shows that reductive alkylation of the enzyme
did not result in any significant changes in the position
and hydrogen bond pattern of active site residues and the
PLP co-factor. Moreover, the hinge angle between N- and
C-terminal domains is very similar for both modified and
unmodified AlrBax. Thus, the hinge angles observed for
AlrBax are inherent to this particular enzyme and not an
artifact of crystallization. As an aside, this observation
makes a strong argument that the disparate hinge angles
observed for other Alrs are not a consequence of divergent
Reductive methylation also did not significantly alter the
dimer interface, which is found to be comparable between
methylated and unmethylated structures (3600 2 vs.
3500 2, respectively). For the modified structure, two
methylated lysines contribute atoms from their methyl
groups to the interface; Mly182 and Mly255. The
corresponding lysines in the native structure are not considered
to be part of the interface; Lys182 displays poor density
and did not have its complete side chain modeled in the
native structure and no atoms from Lys255 in one
monomer are in contact distance to atoms in the other
Only two lysines escaped methylation in the modified
crystal structure, Lys41 and Lys260 . Lys41 is found
covalently bound to the PLP co-factor. Thus its NH group
is not a primary amine and is not surprising that this
residue is unaffected by the reductive methylation protocol.
Lys260 is the lysine residue least exposed to the solvent
and it makes hydrogen bonds to Gly137 and Arg138
which, in turn, hydrogen bonds to the phenolic oxygen of
the PLP cofactor and to the substrate (see above). These
two residues are, therefore, involved in either a covalent
bond or a strong polar interaction in the present structure
and thus predictably escaped reductive methylation.
In conclusion, we report the high-resolution crystal
structure of alanine racemase from the dal1 gene of B. anthracis
and characterize it kinetically and in an E. coli
complementation system. This structure contains some unique
features in its active site including a structural chloride
atom. It shares a similar hinge angle to its close relative
from Geobacillus and has an active site and topology much
like other members of this family. Based on the results
shown here the active site of AlrBax is as accessible for
inhibitor binding as other alanine racemases studied to
date. Furthermore, it is very likely that alanine racemase
inhibitors like D-cycloserine or alanine phosphonate will
be effective as modulators of sporulation. Finally, as
treatment of spores will take place in the environment and not
internally, the problems associated with non-specific PLP
inhibition ascribed to these inhibitors should not detract
from their usefulness in bioremediation. We look forward
to exploring more structural studies on these inhibitors as
they become available.
Amplification and cloning of the B. anthracis alr genes
Two putative open reading frames, dal1 and dal2, for
alanine racemase from B. anthracis were identified
through sequence comparisons using the known alanine
racemase sequence from G. stearothermophilus  as a
probe against the B. anthracis genome deposited in
GenBank . Two sets of primers were used in PCR to
amplify the two putative alr genes from genomic DNA of
B. anthracis (Ames), dal15' (5'-GGG GCC ATG GAA GAA
GCA CCA TTT TAT CGT G-3')/dal13' (5'-CCC CCT CGA
GTA TAT CGT TCA AAT AAT TAA TTA C-3') and dal25'
(5'-GGG GCA TAT GAG TTT GAA ATA TGG AAG AG-3')/
dal23' (5' CCCCCTGCAGAATCCGTAGGTTTTAAGGAC
3'), resulting in amplicons of 1169 bp and 1175 bp,
respectively. The PCR products were sequenced, inserted
into a modified pET28 vector (pET28-TEV) containing a
C-terminal His-tag and a TEV protease cleavage sequence,
LEENLYFQ/SLQVEH6 and cloned in E. coli MB1547. (/)
denotes the location of the cleavage site.
Characterization of the two cloned genes continued with
their transformation into the D-alanine auxotrophic E.
coli strain MB2795 . A plasmid encoding the cloned P.
aeruginosa DadX alanine racemase, pMB1921 , was
used as a positive control. Plasmid pET28-TEV without
any inserts served as the negative control. Cells were
grown on solid LB medium with and without D-alanine
supplementation, and scored for colony growth after 16 h
at 37C as described previously .
Dal1 overexpression and purification
Cultures of E. coli BL21(DE3), pLysS containing the
pET28-TEV-dal expression plasmids were grown at 37C
in LB medium containing 100 g/ml kanamycin and 30
g/ml chloramphenicol to an OD600 of 0.8. Expression of
recombinant proteins was induced by addition of 0.5 mM
IPTG and carried at 30C for 19 hours. Cells were
harvested by centrifugation and the cell lysate was cleared
and loaded onto a Hi Trap affinity (Ni2+) column (GE
Healthcare Life Sciences). The column was washed and
AlrBax eluted with a stepwise imidazole gradient. The
Cterminal 6xHis tag was removed by treatment with
Histagged TEV protease (1 mg TEV protease per 10 mg of
protein for 16 hours at 4C). AlrBax without the 6xHis tag was
purified from the reaction mixture using the same
chromatography strategy described above. Following
concentration, AlrBax was loaded onto a Pharmacia Superdex 200
Preparative Grade column; sample purity was assessed by
SDS-PAGE to be greater than 95%.
Dynamic light scattering
Purified AlrBax was dialyzed against 20 mM Tris pH 8.0.
Protein samples (1 mg/ml) were centrifuged (10 min. at
14,000 rpm) and filtered using 0.02 m Whatman
Anotop filters prior to recording data. All measurements
were made at 298 K using the DynaPro system according
to the manufacturer's instructions (Wyatt Technology).
Enzyme Kinetics and Crystallization
The kinetic parameters (Km and Vmax) for the racemization
reaction (D- to L-alanine) catalyzed by AlrBax were
estimated using the spectrophotometric alanine racemase
assay as described previously . AlrBax crystallization
screening trials were performed using the vapor diffusion
method with sitting drops (5 l of protein at 15 mg/ml
and 5 l of mother liquor) in 24-well plates incubated at
4C. Initial screens revealed thin needle crystals growing
in 20% PEG 8000, 0.2 M sodium acetate, 0.1 M sodium
cacodylate, pH 6.5 . Crystals were optimized using
streak-seeding with crushed crystals and further
optimized using additive screening resulting in rectangular,
deep yellow crystals suitable for data collection. The final
crystallization condition was 18% PEG 8000, 0.2 M
sodium acetate, 0.1 M sodium cacodylate, pH 6.5, 0.01 M
GSH (L-glutathione reduced), 0.01 M GSSG
Data Collection and Processing
Crystals were passed through cryoprotectant solutions
consisting of 20.7% PEG 8000, 0.2 M sodium acetate, 0.1
M sodium cacodylate supplemented with 3, 6, 9, 12, 15
and 18% (v/v) ethylene glycol, mounted into a nylon
loop and flash frozen in liquid nitrogen at 110 K. A native
data set was collected at 110 K on a Micromax 007 HF
rotating-anode X-ray generator equipped with a copper
anode, Hi-res optics, an RAXIS IV++ image-plate detector
(Rikagu) using a frame width of 0.5 and an exposure
time of 600 s. Images were integrated using MOSFLM
, processed with SCALA  and analyzed using
programs from the CCP4 suite . Data collection and
processing statistics for the native data set can be found in
Table 1. AlrBax crystallized in space group P21 with unit cell
parameters a = 49.62 , b = 141.27 , c = 60.12 and =
103.11. There is one AlrBax dimer per asymmetric unit.
Structure Determination and Refinement
Molecular replacement was carried out with MolRep 
using the G. stearothermophilus Alr (PDB entry 1SFT)
atomic coordinates . Molecular replacement was
performed assuming two monomers per asymmetric unit as
suggested by a Matthew's coefficient of 2.35  and
resulted in the proper orientation of the search model in
the crystal lattice (Rfac 43.6%; score 0.699). The primary
sequence of the search model was changed to that of AlrBax
using Coot . All structural refinements (32.79 1.95
) were carried in Refmac5  using standard restraints
and were followed by visual inspection of protein models
and density maps in Coot. Ten cycles of positional
refinement, performed using NCS restraints, resulted in R and
Rfree of 23.9 and 27.2%, respectively. Waters were added
using the arp_water function on Refmac5, and when the
active site density was clearly interpretable, PLP was added
to both active sites. A further 10 cycles of positional and
Biso refinements brought R and Rfree to 19.6 and 23.7%,
respectively. Water molecules with B-factors higher than
55.0 2 and electron density lower than 1.0 on a 2Fobs
Fcalc map were then deleted.
B. anthracis crystals displayed somewhat anisotropic x-ray
diffraction and previous alanine racemase structures have
shown indication of subdomain movement. This
encouraged us to try TLS refinement . TLS analyses were
carried on with different domains of the protein acting as a
rigid body. All models resulted in similar improvements
in R and Rfree and in the end we adopted the most
parsimonious one, which treated all protein atoms found in
the asymmetric unit as a rigid body. After TLS refinement,
the R and Rfree were 16.0 and 20.1% with
root-meansquare deviations from ideality for bond lengths of 0.017
and angles of 1.46 (Table 1). As noted above, inclusion
of the C-terminal His-tag has resulted in eight additional
residues in our sequence. In the final map we attempted
to build some of these residues into extra density at the
Cterminus, but as we did not gain anything in terms of R or
Rfree we have elected to leave out the extra residues from
this region in the final structure. Structure factors and final
atomic coordinates for AlrBax have been deposited in the
Protein Databank (PDB ID 3ha1).
The structure of AlrBax was compared to other closely
related enzymes; their accession numbers are: 1sft AlrGst
bound with acetate ; 1vfh AlrSla with no ligand ;
2vd8 methylated AlrBax , 1rcq DadXPao  and
1xfc AlrMtb . Structural alignments were performed
using SSM . Interface surface area was calculated using
PISA . The number of polar contacts (hydrogen bonds
and salt bridges) was determined using WHAT IF [61,62].
List of Abbreviations
Alr: alanine racemase; Bax: Bacillus anthracis; DCS:
Dcycloserine; Gst: Geobacillus stearothermophilus; Mtb:
Mycobacterium tuberculosis; Pao: Pseudomonas aeruginosa; PLP:
pyridoxal 5'-phosphate; rms: root mean square; Sla:
RC performed research, helped draft manuscript,
analyzed results, and prepared figures. US performed
research, helped draft manuscript, analyzed results. MD
performed research, helped draft manuscript, and
analyzed results. RH helped analyze structure and helped
prepare figures. KK designed research, analyzed results,
helped draft manuscript. All authors read and approved
the final manuscript.
This work was supported by funding from the University of Otago, the
Robert A. Welch Foundation, the National Institutes of Health, the Thrash
Foundation and the Foundation for the Centers for Molecular Research in
Infectious Diseases. We would like to dedicate this manuscript to John
Francis Thrash, MD, a noted philanthropist, physician and businessman
from Houston, Texas who generously aided in the establishment of our
structural biology laboratory in New Zealand.
1. Dixon TC , Meselson M , Guillemin J , Hanna PC : Anthrax . N Engl J Med 1999 , 341 ( 11 ): 815 - 826 .
2. Dahlgren CM , Buchanan LM , Decker HM , Freed SW , Phillips CR , Brachman PS : Bacillus anthracis aerosols in goat hair processing mills . Am J Hyg 1960 , 72 : 24 - 31 .
3. Meselson M , Guillemin J , Hugh-Jones M , Langmuir A , Popova I , Shelokov A , Yampolskaya O : The Sverdlovsk anthrax outbreak of 1979 . Science 1994 , 266 ( 5188 ): 1202 - 1208 .
4. CDC: Use of anthrax vaccine in the United States: recommendations of the Advisory Committee on Immunization Practices (ACIP) . MMWR 2000 , 49 ( RR15 ): 1 - 20 .
5. Radosavljevic V , Jakovljevic B : Bioterrorism - types of epidemics, new epidemiological paradigm and levels of prevention . Public Health 2007 , 121 ( 7 ): 549 - 557 .
6. Jernigan JA , Stephens DS , Ashford DA , Omenaca C , Topiel MS , Galbraith M , Tapper M , Fisk TL , Zaki S , Popovic T , et al.: Bioterrorismrelated inhalational anthrax: the first 10 cases reported in the United States . Emerg Infect Dis 2001 , 7 ( 6 ): 933 - 944 .
7. Read TD , Salzberg SL , Pop M , Shumway M , Umayam L , Jiang L , Holtzapple E , Busch JD , Smith KL , Schupp JM , et al.: Comparative genome sequencing for discovery of novel polymorphisms in Bacillus anthracis . Science 2002 , 296 ( 5575 ): 2028 - 2033 .
8. Lambert MP , Neuhaus FC : Mechanism of D-cycloserine action: alanine racemase from Escherichia coli W . J Bacteriol 1972 , 110 ( 3 ): 978 - 987 .
9. Silverman RB : The potential use of mechanism-based enzyme inactivators in medicine . J Enzyme Inhib 1988 , 2 ( 2 ): 73 - 90 .
10. Todd SJ , Moir AJ , Johnson MJ , Moir A : Genes of Bacillus cereus and Bacillus anthracis encoding proteins of the exosporium . J Bacteriol 2003 , 185 ( 11 ): 3373 - 3378 .
11. Titball RW , Manchee RJ : Factors affecting the germination of spores of Bacillus anthracis . J Appl Bacteriol 1987 , 62 ( 3 ): 269 - 273 .
12. McKevitt MT , Bryant KM , Shakir SM , Larabee JL , Blanke SR , Lovchik J , Lyons CR , Ballard JD : Effects of endogenous D-alanine synthesis and autoinhibition of Bacillus anthracis germination on in vitro and in vivo infections . Infect Immun 2007 , 75 ( 12 ): 5726 - 5734 .
13. Delvecchio VG , Connolly JP , Alefantis TG , Walz A , Quan MA , Patra G , Ashton JM , Whittington JT , Chafin RD , Liang X , et al.: Proteomic profiling and identification of immunodominant spore antigens of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis . Appl Environ Microbiol 2006 , 72 ( 9 ): 6355 - 6363 .
14. Huang CM , Foster KW , DeSilva TS , Van Kampen KR , Elmets CA , Tang DC : Identification of Bacillus anthracis proteins associated with germination and early outgrowth by proteomic profiling of anthrax spores . Proteomics 2004 , 4 ( 9 ): 2653 - 2661 .
15. Veerapandian B : Three Dimensional Structure-Aided Drug Design . In Burger's Medicinal Chemistry and Drug Discovery Volume 1. 5th edition. Edited by: Wolff ME. New York, New York : John Wiley & Sons, Inc.; 1995 : 303 - 348 .
16. Marrone TJ , Briggs JM , McCammon JA : Structure-based drug design: computational advances . Annu Rev Pharmacol Toxicol 1997 , 37 : 71 - 90 .
17. Blundell TL : Structure-based drug design . Nature 1996 , 384 (Supp): 23 - 26 .
18. Blythe MJ , Flower DR : Benchmarking B cell epitope prediction: underperformance of existing methods . Protein Sci 2005 , 14 ( 1 ): 246 - 248 .
19. Pizarro JC , Vulliez-Le Normand B , Chesne-Seck ML , Collins CR , Withers-Martinez C , Hackett F , Blackman MJ , Faber BW , Remarque EJ , Kocken CH , et al.: Crystal structure of the malaria vaccine candidate apical membrane antigen 1 . Science 2005 , 308 ( 5720 ): 408 - 411 .
20. Tolia NH , Enemark EJ , Sim BK , Joshua-Tor L : Structural basis for the EBA-175 erythrocyte invasion pathway of the malaria parasite Plasmodium falciparum . Cell 2005 , 122 ( 2 ): 183 - 193 .
21. Morollo AA , Petsko GA , Ringe D : Structure of a Michaelis complex analogue: propionate binds in the substrate carboxylate site of alanine racemase . Biochemistry 1999 , 38 ( 11 ): 3293 - 3301 .
22. Shaw JP , Petsko GA , Ringe D : Determination of the structure of alanine racemase from Bacillus stearothermophilus at 1.9 resolution . Biochemistry 1997 , 36 ( 6 ): 1329 - 1342 .
23. LeMagueres P , Im H , Dvorak A , Strych U , Benedik M , Krause KL : Crystal structure at 1.45 resolution of alanine racemase from a pathogenic bacterium, Pseudomonas aeruginosa, contains both internal and external aldimine forms . Biochemistry 2003 , 42 ( 50 ): 14752 - 14761 .
24. Fenn TD , Stamper GF , Morollo AA , Ringe D : A side reaction of alanine racemase: transamination of cycloserine . Biochemistry 2003 , 42 ( 19 ): 5775 - 5783 .
25. Stamper GF , Morollo AA , Ringe D : Reaction of alanine racemase with 1-aminoethylphosphonic acid forms a stable external aldimine . Biochemistry 1998 , 37 ( 29 ): 10438 - 10445 .
26. LeMagueres P , Im H , Ebalunode J , Strych U , Benedik MJ , Briggs JM , Kohn H , Krause KL : The 1.9 crystal structure of alanine racemase from Mycobacterium tuberculosis contains a conserved entryway into the active site . Biochemistry 2005 , 44 ( 5 ): 1471 - 1481 .
27. Noda M , Matoba Y , Kumagai T , Sugiyama M : Structural evidence that alanine racemase from a D-cycloserine-producing microorganism exhibits resistance to its own product . J Biol Chem 2004 , 279 ( 44 ): 46153 - 46161 .
28. Au K , Ren J , Walter TS , Harlos K , Nettleship JE , Owens RJ , Stuart DI , Esnouf RM : Structures of an alanine racemase from Bacillus anthracis (BA0252) in the presence and absence of (R)-1-aminoethylphosphonic acid (L-Ala-P) . Acta Crystallogr Sect F Struct Biol Cryst Commun 2008 , 64 (Pt 5): 327 - 333 .
29. Kobayashi M , Kubota M , Matsuura Y : Crystallization and improvement of crystal quality for x-ray diffraction of maltooligosyl trehalose synthase by reductive methylation of lysine residues . Acta Crystallogr D Biol Crystallogr 1999 , 55 (Pt 4): 931 - 933 .
30. Kurinov IV , Mao C , Irvin JD , Uckun FM : X-ray crystallographic analysis of pokeweed antiviral protein-II after reductive methylation of lysine residues . Biochem Biophys Res Commun 2000 , 275 ( 2 ): 549 - 552 .
31. Rayment I , Rypniewski WR , Schmidt-Base K , Smith R , Tomchick DR , Benning MM , Winkelmann DA , Wesenberg G , Holden HM : Threedimensional structure of myosin subfragment-1: a molecular motor . Science 1993 , 261 ( 5117 ): 50 - 58 .
32. Saxena AK , Singh K , Su HP , Klein MM , Stowers AW , Saul AJ , Long CA , Garboczi DN : The essential mosquito-stage P25 and P28 proteins from Plasmodium form tile-like triangular prisms . Nat Struct Mol Biol 2006 , 13 ( 1 ): 90 - 91 .
33. Schubot FD , Waugh DS : A pivotal role for reductive methylation in the de novo crystallization of a ternary complex composed of Yersinia pestis virulence factors YopN, SycN and YscB . Acta Crystallogr D Biol Crystallogr 2004 , 60 (Pt 11): 1981 - 1986 .
34. Walter TS , Meier C , Assenberg R , Au KF , Ren J , Verma A , Nettleship JE , Owens RJ , Stuart DI , Grimes JM : Lysine methylation as a routine rescue strategy for protein crystallization . Structure 2006 , 14 ( 11 ): 1617 - 1622 .
35. Kim Y , Quartey P , Li H , Volkart L , Hatzos C , Chang C , Nocek B , Cuff M , Osipiuk J , Tan K , et al.: Large-scale evaluation of protein reductive methylation for improving protein crystallization . Nat Methods 2008 , 5 ( 10 ): 853 - 854 .
36. Read TD , Peterson SN , Tourasse N , Baillie LW , Paulsen IT , Nelson KE , Tettelin H , Fouts DE , Eisen JA , Gill SR , et al.: The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria . Nature 2003 , 423 ( 6935 ): 81 - 86 .
37. Huang CM , Elmets CA , Tang DC , Li F , Yusuf N : Proteomics reveals that proteins expressed during the early stage of Bacillus anthracis infection are potential targets for the development of vaccines and drugs . Genomics Proteomics Bioinformatics 2004 , 2 ( 3 ): 143 - 151 .
38. Strych U , Penland RL , Jimenez M , Krause KL , Benedik MJ : Characterization of the alanine racemases from two mycobacteria . FEMS Microbiol Lett 2001 , 196 ( 2 ): 93 - 98 .
39. Inagaki K , Tanizawa K , Badet B , Walsh CT , Tanaka H , Soda K : Thermostable alanine racemase from Bacillus stearothermophilus: molecular cloning of the gene , enzyme purification, and characterization. Biochemistry 1986 , 25 ( 11 ): 3268 - 3274 .
40. Strych U , Huang HC , Krause KL , Benedik MJ : Characterization of the alanine racemases from Pseudomonas aeruginosa PAO1 . Curr Microbiol 2000 , 41 ( 4 ): 290 - 294 .
41. Strych U , Benedik MJ : Mutant analysis shows that alanine racemases from Pseudomonas aeruginosa and Escherichia coli are dimeric . J Bacteriol 2002 , 184 ( 15 ): 4321 - 4325 .
42. Boggetto N , Reboud-Ravaux M : Dimeric Inhibitors of HIV -1 Protease . Biol Chem 2002 , 383 ( 9 ): 1321 - 1324 .
43. Song M , Rajesh S , Hayashi Y , Kiso Y : Design and Synthesis of New Inhibitors of HIV-1 Protease Dimerization with Conformationally Constrained Templates . Bioorg Med Chem Lett 2001 , 11 ( 18 ): 2465 - 2468 .
44. Strosberg AD : Breaking the spell: drug discovery based on modulating protein-protein interactions . Expert Rev Proteomics 2004 , 1 ( 2 ): 141 - 143 .
45. Wu D , Hu T , Zhang L , Chen J , Du J , Ding J , Jiang H , Shen X : Residues Asp164 and Glu165 at the substrate entryway function potently in substrate orientation of alanine racemase from E. coli: Enzymatic characterization with crystal structure analysis . Protein Sci 2008 , 17 ( 6 ): 1066 - 1076 .
46. Au K , Berrow NS , Blagova E , Boucher IW , Boyle MP , Brannigan JA , Carter LG , Dierks T , Folkers G , Grenha R , et al.: Application of high-throughput technologies to a structural proteomicstype analysis of Bacillus anthracis . Acta Crystallogr D Biol Crystallogr 2006 , 62 (Pt 10): 1267 - 1275 .
47. Rauert W , Eddine AN , Kaufmann SH , Weiss MS , Janowski R : Reductive methylation to improve crystallization of the putative oxidoreductase Rv0765c from Mycobacterium tuberculosis . Acta Crystallogr Sect F Struct Biol Cryst Commun 2007 , 63 (Pt 6): 507 - 511 .
48. Rypniewski WR , Holden HM , Rayment I : Structural consequences of reductive methylation of lysine residues in hen egg white lysozyme: an X-ray analysis at 1.8-A resolution . Biochemistry 1993 , 32 ( 37 ): 9851 - 9858 .
49. Strych U , Davlieva M , Longtin JP , Murphy EL , Im H , Benedik MJ , Krause KL : Purification and preliminary crystallization of alanine racemase from Streptococcus pneumoniae . BMC Microbiol 2007 , 7 : 40 .
50. Jancarik J , Kim S-H : Sparse matrix sampling: a screening method for crystallization of proteins . J Appl Crystallogr 1991 , 24 : 409 - 411 .
51. Leslie AGW : Recent changes to the MOSFLM package for processing film and image plate data . Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography 1992 , 26 : 27 - 33 .
52. Evans PR : SCALA, version 3 .1.9. Medical Research Council Laboratory of Molecular Biology , Cambridge, UK 1993 .
53. CCP4 suite: The CCP4 suite: programs for protein crystallography . Acta Crystallogr D Biol Crystallogr 1994 , 50 (Pt 5): 760 - 763 .
54. Vagin A , Teplyakov A : MOLREP: an automated program for molecular replacement . J Appl Cryst 1997 , 30 : 1022 - 1025 .
55. Matthews BW : Solvent Content of Protein Crystals . J Mol Biol 1968 , 33 : 491 - 497 .
56. Emsley P , Cowtan K : Coot: model-building tools for molecular graphics . Acta Crystallogr D Biol Crystallogr 2004 , 60 (Pt 12 Pt 1): 2126 - 2132 .
57. Murshudov GN , Vagin AA , Dodson EJ : Refinement of macromolecular structures by the maximum-likelihood method . Acta Crystallogr D Biol Crystallogr 1997 , 53 (Pt 3): 240 - 255 .
58. Schomaker V , Trueblood KN : On the rigid-body motion of molecules in crystals . Acta Cryst, Section B 1968 , 24 ( 1 ): 63 - 76 .
59. Krissinel E , Henrick K : Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions . Acta Crystallogr D Biol Crystallogr 2004 , 60 (Pt 12 Pt 1): 2256 - 2268 .
60. Krissinel E , Henrick K : Inference of macromolecular assemblies from crystalline state . J Mol Biol 2007 , 372 ( 3 ): 774 - 797 .
61. Vriend G : WHAT IF: a molecular modeling and drug design program . J Mol Graph 1990 , 8 ( 1 ): 52 - 56 . 29.
62. Rodriguez R , Chinea G , Lopez N , Pons T , Vriend G : Homology modeling, model and software evaluation: three related resources . CABIOS 1998 , 14 : 523 - 528 .
63. O'Sullivan O , Suhre K , Abergel C , Higgins DG , Notredame C : 3DCoffee: combining protein sequences and structures within multiple sequence alignments . J Mol Biol 2004 , 340 ( 2 ): 385 - 395 .