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INTRODUCTION |
The final step in the bacterial lysine biosynthetic pathway is
carried-out by
meso-DAP1
decarboxylase (DAPDC), encoded by the lysA gene. DAPDC is a
vitamin B6-dependent enzyme that
stereospecifically converts meso-DAP to L-lysine
(Scheme 1). Like most enzyme-catalyzed decarboxylation reactions, the
conversion of DAP to lysine is not reversible. The enzyme is of
interest because of its importance in bacterial growth and survival.
Lysine is required in protein biosynthesis and is essential for
bacterial viability and development. The lysine precursor DAP itself
is used as a structural cross-linking component of the peptidoglycan
layer of Gram-negative, Gram-positive (except Gram-positive
cocci), and mycobacterial cell walls (1). DAP cross-links
provide stability to the cell wall and confer resistance to
intracellular osmotic pressure (2). DAP can be synthesized by one or
more of the following three different pathways: (i) the succinylase
pathway, identified in all Gram-negative and Gram-positive bacteria, as
well as Myobacterium tuberculosis; (ii) the dehydrogenase
pathway, utilized by Bacillus sphaericus, Corynebacterium
glutamicum, and Brevibacterium species (3); and (iii)
the acetylase pathway, which is limited to certain Bacillus species (4). Higher plants also produce lysine using a succinylase pathway (5). The presence of multiple biosynthetic pathways, at least
in some bacteria, is probably an indication of the importance of DAP
and lysine to bacterial survival. As the substrate and the reaction are
not found in mammals, inhibitors of the enzyme may ultimately become
leads for therapeutic intervention in bacterial infections (6).
In Escherichia coli, the lysA gene is
transcriptionally controlled by the LysR regulator protein; in the
presence of lysine, transcription of the lysA gene is
repressed (7). In contrast, M. tuberculosis does not
apparently have a comparable LysR regulator, based on the lack of
homologous sequences in the M. tuberculosis genomic
sequence (8). In M. tuberculosis, C. glutamicum,
and Brevibacterium lactofermentum, the lysA gene
is not in an operon as the second gene in an open reading frame with
argS (arginyl-tRNA synthetase) (9-12). In C. glutamicum the lysA gene is constitutively expressed
(11), and in the related organism B. lactofermentum the
lysA gene is only weakly suppressed by lysine (12). Based on
the evolutionary relationship between these three species of bacteria,
we (13) proposed that the expression of the lysA gene of
M. tuberculosis is probably constitutive.
We show in this study that the lysA gene is essential for
M. tuberculosis survival in an immunodeficient SCID (severe
combined immunodeficient) mouse model, and we have determined the
crystal structure of DAPDC in complex with the coenzyme pyridoxal
5'-phosphate (PLP) and the decarboxylation product lysine as well as
DAPDC complexed with only lysine (binary complex). DAPDC is
structurally very similar to eukaryotic ornithine decarboxylases (ODCs)
(14-16) and, with the exception of a rotation of the C-terminal
domain, to Bacillus stearothermophilus alanine racemase (AR)
(17). Although both DAPDC and ODCs carry-out similar decarboxylation
reactions involving pyridoxal-5'-phosphate (PLP) as a cofactor, DAPDC
is the only known amino acid decarboxylase that stereospecifically acts
on a substrate carbon atom in D-configuration (Scheme
1).

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Scheme 1.
Reaction schematic of stereospecific
decarboxylation of meso-diaminopimelic acid (DAP) to
L-lysine via vitamin B6
(PLP)-dependent DAP-decarboxylase (DAPDC).
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EXPERIMENTAL PROCEDURES |
Generation and in Vitro Characterization of the lysA Mutant of
M. tuberculosis--
The lysA mutant of M. tuberculosis, mc23026, was previously constructed by
allelic exchange and has a deletion within the coding region of the
lysA gene with an inserted 
resolvase binding site
(18). The mutant requires exogenous lysine supplementation at 1 mg/ml
and can be complemented to protrophy by a copy of the wild-type
lysA gene carried on the integrating vector pYUB651. In this
work, we performed reversion analysis and were unable to isolate
revertants from over 1010 M. tuberculosis
lysA cells. This established that the DAPDC activity can
not be suppressed by any extragenic mutation and that the viability of
the M. tuberculosis cells is dependent on this activity.
Clearance of the M. tuberculosis Lysine Auxotroph in
SCID Mice--
Female SCID mice were bred at the animal facility of
the Albert Einstein College of Medicine. The animals were maintained under barrier conditions and fed sterilized commercial mouse chow and
water ad libitum. The M. tuberculosis
strains mc23026 (
lysA5::res) and
mc23026 bearing pYUB651 (expressing the wild-type
lysA gene) (13), were grown in Middlebrook 7H9 broth (Difco)
supplemented with 0.05% Tween 80, 0.2% glycerol, and 1× ADS (0.5%
bovine serum albumin, fraction V (Roche), 0.2% dextrose, and 0.85%
NaCl) or on Middlebrook 7H10 or 7H11 solid medium (Difco) supplemented
with 0.2% glycerol and 10% OADC (oleic acid, albumin, dextrose, and
catalase; BD Biosciences). Cultures of the lysine auxotroph were
supplemented with 1 mg/ml L-lysine (for both liquid and
solid media), and 0.05% Tween 80 was added to solid medium. Liquid
cultures were grown in 490-cm2 roller bottles (Corning) at
4-6 rpm. Plates were incubated for 3-6 weeks.
Titered frozen stocks of bacteria were thawed and diluted appropriately
in phosphate buffered saline containing 0.05% Tween 80 (PBST). The
bacterial suspensions were plated at the time of injection to confirm
viable counts. Intravenous injections were given via the tail vein. At
various time points post-injection (24 h and once weekly), three mice
were sacrificed for each strain, and the lungs, liver, and spleen were
removed and homogenized separately in PBST using a Stomacher 80 (Tekmar, Cincinnati, OH). The homogenates were diluted in PBST and
plated to determine the number of colony-forming units (CFU)/ml. Note
that mice were sacrificed at 24 h post-injection in order to
compare the bacterial colony-forming units received by the mice to the
colony-forming units in the suspensions at the time of injection. Thus,
the bacterial counts reported at time zero represent the viable
bacteria present in the mice at 24 h post-injection.
Cloning of the lysA Gene and Expression of M. tuberculosis
DAPDC--
A 1.3-kb DNA fragment containing the
lysA gene (Rv1237, Swiss Prot accession number
P31848), was amplified by PCR with M. tuberculosis H37Rv genomic DNA as the
template, using the following oligonucleotide primers: 5'-AGA
GAA GCA TAT GAA CGA GCT GCT GCA CTT AGC GCC GAA TG-3' and 5'-AGA GAA
GGC GGC CGC CCT CAC TTC CAA ACT CAG CAA ATC GTC-3'. The amplified DNA
fragment was digested with NdeI and NotI
restriction enzymes and subcloned into the corresponding restriction
sites in the pET30b vector with a C-terminal His6 tag.
E. coli B834 (DE3) Met
cells were transformed
with the lysA-pET30b/His vector. The transformed cells were grown to
exponential phase at 37 °C in TB media containing kanamycin. For
production of Se-Met labeled protein, the cells were grown in M9
minimal media supplemented with all 19 standard amino acids and
selenium-methionine (19). Expression of lysA was induced
with 1 mM
isopropyl-1-thio-
-D-galactopyranoside (IPTG), and cells
were harvested after growth for 4-6 h at 16 °C.
DAPDC Purification--
The harvested cells were pelleted and
resuspended in buffer A (20 mM Tris-HCl, pH 8.0, and 50 mM imidazole) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and complete EDTA-free
protease inhibitors (Roche Applied Science). The cell mixture was
repeatedly sonicated at 4 °C with 30 s pulses, and the cell
suspension was centrifuged at 15,000 × g for 1 h.
The clear supernatant was loaded onto an Amersham Biosciences
Hi-trap Ni2+ chelating column and washed with 300 ml of
buffer A containing 500 mM NaCl. The His-tagged DAPDC was
eluted from the nickel affinity column using Buffer B (20 mM Tris-HCl, pH8.0, 500 mM imidazole, and 500 mM NaCl). After purification to near homogeneity by size exclusion chromatography (Amersham Biosciences) on an S-Superdex-200 column, DAPDC was dialyzed against 20 mM Tris buffer (pH
8.0), concentrated to 10 mg/ml, and stored in 20 mM
Tris-HCl, pH 8.0, at
80 °C.
Crystallization--
Native and Se-Met-labeled DAPDC (10 mg
ml
1) were crystallized at 18 °C by vapor diffusion in
hanging drops. Initial crystallization screening was carried out with
DAPDC alone, DAPDC incubated with DAP (5 mM) plus PLP (0.2 mM) overnight at 4 °C, and DAPDC plus lysine.
Crystallization of DAPDC was only successful in the case of DAPDC
supplemented with 5 mM lysine. Crystals (0.2 × 0.3 × 0.3 mm) grew at 18 °C within 3-7 days in 4-µl hanging
drops (2 µl of DAPDC, 10 mg/ml, containing 5 mM of lysine
combined with 2 µl of well solution) equilibrated against 500 µl of
well solution containing 24% polyethylene glycol mono-methylether 5000 (PEG-MME 5000), 0.1 M MES buffer, pH 6.3, and 60 mM ammonium sulfate. Native DAPDC-lysine crystals were
soaked for 3 h in mother liquor containing 0.2 mM PLP
to obtain distinctly yellow-colored crystals of the DAPDC-PLP-lysine complex.
Data Collection--
Highly redundant and complete selenium
K-edge MAD diffraction data from a single Se-Met-DAPDC/lysine crystal
were collected at three wavelengths using an ADSC CCD detector on
beamline 14-ID-B at the Advanced Photon Source (APS) of the Argonne
National Laboratory (ANL). Crystals mounted in cryo-loops were flash
cooled in a N2 stream (120 K) after brief soaks in 2 µl
of mother liquor plus 2 µl of a cryoprotectant composed of 30%
dioxane and 20% 2-methyl-2,4-pentanediol (MPD). Native data
from DAPDC-PLP-lysine crystals were recorded on APS beamline 19BM using
the 3 × 3 segment APS-1 CCD detector. The diffraction data were
reduced using DENZO (20), and intensities were scaled with SCALEPACK
(20). The reflections were indexed primitive tetragonal (a = b = 111.5 Å, c = 237.7 Å) with Laue symmetry 4/mmm.
Examination of the integrated and scaled data indicated tetragonal
space group P41212 or its
enantiomorph P43212. Solvent content
calculations (21) indicated the presence of either a dimer
(VM, 4.0; VS, 70%) or a trimer
(VM, 2.8; VS, 54%) in the asymmetric unit.
Structure Determination--
Experimental phases for
DAPDC-lysine were obtained by multiwavelength anomalous diffraction
(MAD) phasing (22) (Table I). SHELXD located eight selenium sites in the asymmetric unit consistent with a dimer in the asymmetric unit (23), and SOLVE (24) was used to
refine the sites and calculate initial protein phases, resulting in an
overall figure of merit of 0.41 for the data in the resolution range of
100-2.8 Å. Further phase improvement with solvent flattening in
AUTOSHARP (25) resulted in density-modified maps of high quality
showing clear electron density for two molecules of protein in the
asymmetric unit. The electron density map was submitted to TEXTAL (26)
for automated model building. The TEXTAL model fit 80% of the backbone
and 20% of the side chains correctly, with the exception of a stretch
of 50 amino acids that were traced in the wrong direction; the
remaining backbone model fit well into the electron density of the map.
After determining the non-crystallographic symmetry (NCS)
operator from the selenium substructure using graphical analysis and
refinement with (CCP4) LSQKAB, the electron density was averaged and
solvent flattened using DM (27). Starting from the TEXAL tracing, all
of the residues of DAPDC except Met-1 could be built into the
density-modified and -averaged experimental map using XTALVIEW (28). A
final model of high quality was produced after several cycles of manual
model building, and NCS restrained maximum likelihood refinement with
REFMAC5 (29) against the high remote data set (Table
II). A sulfate ion, located at the position of the PLP phosphate moiety, was clearly visible in the electron density. 204 water molecules were manually added during iterative cycles of model building and refinement. Weak electron density for the complexed lysine was visible in each binding pocket of
the dimer but was not refined in the Se-Met model.
The structure of native DAPDC complexed with PLP and lysine was solved
by molecular replacement with EPMR (30) (correlation coefficient 0.60)
using the final model of the Se-Met DAPDC-lysine complex as a search
model. Bias-minimized electron density maps were obtained using the
Shake&wARP (SNW) protocol (31). Clear electron density for both PLP
molecules and density for both lysines were visible in the Shake&wARP
map prior to any model building. Several cycles of manual model
adjustment and NCS-restrained maximum likelihood refinement in REFMAC5
yielded a final 2.6 Å model of good quality (Table II) for the
DAPDC-PLP-lysine complex.
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RESULTS AND DISCUSSION |
The lysA Gene Is Required for in Vivo Growth of M. tuberculosis
H37Rv--
The lysine auxotrophic strain mc23026 or the
complemented mutant were each introduced (106 cells per
mouse) into 24 SCID mice by tail vein injections, and groups of three
mice each were sacrificed at 1 day post-injection and weekly thereafter
until week 6. At each sacrifice, the number of viable bacteria was
determined in the spleens, livers, and lungs of the mice. The lysine
auxotrophic mutant was cleared from or did not grow in the examined
organs of the SCID mice, whereas the complemented strain,
mc23026/pYUB651, multiplied extensively (Fig.
1). In both the spleen and the lung, the
number of viable bacteria decreased by three orders of magnitude in 6 weeks (Fig. 1B), whereas the decrease of the number of
viable bacteria in the lung was only one order of magnitude (Fig.
1C). The mice given the complemented M. tuberculosis mutant died within 3 weeks, whereas the mice
receiving the auxotrophic M. tuberculosis mutant did not
display any gross organ pathology and survived for the duration of the
experiment. Control experiments have demonstrated that immunocompetent
C57BL/6 mice can clear an infection with the M. tuberculosis
lysine auxotroph with the same kinetics as those seen for the clearance
of the mutant in the spleen and lungs of the SCID mice (data not
shown).

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Fig. 1.
Clearance of the lysine auxotrophs in SCID
mice. The viable bacterial counts in CFU/ml are shown for the
spleens, livers, and lungs of SCID mice injected intravenously with the
various mycobacterial strains. Three mice were assayed at each time
point. The error bars indicate the means ± S.D. Note that the counts at time zero are the counts obtained at
24 h post-injection as described under "Results and
Discussion." Panels A, B, and C show
the CFU/ml in each organ after injection with 1 × 107
CFU of the Lys M. tuberculosis mutant
mc23026 (open squares), or 1 × 107 CFU of the complemented Lys+ M. tuberculosis strain mc23026/pYUB651 (closed
squares).
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In addition, we tested the frequency of reversion of the
lysA mutations by growing the mutant in the presence of
lysine to mid-log phase of growth, centrifuging it, and resuspending it in media without lysine. The plating of two independent cultures and
plating over 1010 cells from both cultures yielded no
viable colonies, thus establishing that the lysA deletion mutant does
not revert and cannot be suppressed by an extragenic mutation. The
combination of the in vitro and in vivo data
establishes that DAPDC activity is essential for the viability of
M. tuberculosis and that M. tuberculosis cannot sequester lysine from a mammalian host. We thus reasoned that drugs
targeted against DAPDC could be effective anti-tuberculosis agents and
pursued the determination of the three-dimensional structure of
M. tuberculosis DAPDC.
Overview of the M. tuberculosis DAPDC Structure--
The crystal
structure of M. tuberculosis DAPDC confirms its classification as a
fold type III B6 dependent enzyme (32). DAPDC has a fold
similar to eukaryotic ODCs (14-16), and DAPDC also forms a stable
head-to-tail homodimer of practically identical subunits with a
coordinate deviation comparable with the overall r.m.s.d. coordinate
error for the structure models (0.33 and 0.42 Å, respectively).
Each of the DAPDC subunits (related by proper 2-fold rotation) consists
of two ODC-like domains (Fig. 2). Domain
I is composed of residues 48-308 forming a
/
barrel comprised of
-strands (
4-
13) and helices (
2-
10). The first 47 residues are located in domain II and contain strands
1,
2,
3,
and helix
1, leading into helix
2 of the barrel. The C-terminal
domain II contains residues 2-47 (
1,
2,
3, and
1) and
309-446 (
11-
13, strands
14-
21) and forms a mixed
-sheet flanked by
helices. The two structural domains are
connected by helix
2 and
13. All of the loops connecting the
strands and
helices were clearly visible in the electron
density.

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Fig. 2.
Overview of the M. tuberculosis
DAPDC structure. A, ribbon presentation of
secondary structure elements. The / barrel domain (I) formed of
residues 48-308 is shown in yellow; the C-terminal domain
(II) contains residues 1-47 of the amino-terminal and 309-446 from
the C-terminal region and is colored magenta. The fold is
similar to that of eukaryotic ODCs and classifies M. tuberculosis DAPDC as a fold type III
B6-dependent enzyme. B, two
molecules of DAPDC, related by 2-fold non-crystallographic symmetry,
form a stable dimer. Subunit one, same color scheme as in
panel A. Subunit two is colored cyan
(N-terminal / domain) and red (C-terminal domain).
Shown in stick representation, PLP and lysine, located in
the binding pocket formed by dimer interfaces between N-terminal and
C-terminal domains. Also shown are the disulfide links between the
subunits.
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Two identical binding sites are formed by residues of both polypeptide
chains of the dimer. The active site is at the interface between the
/
barrel domain of one subunit and the
sheet domain of both
subunits. Residues from the
/
barrel are mainly involved in
binding PLP, whereas residues from the
sheet domain primarily contribute to substrate binding. Large conformational changes between
the binary DAPDC-lysine and ternary DAPDC-PLP-lysine complex are absent
(overall C
coordinate r.m.s.d. 0.42 Å). The only significant differences between the DAPDC complex structures appear near the substrate and cofactor binding sites, discussed below.
Comparison of M. tuberculosis DAPDC with Eucaryotic Ornithine
Decarboxylases and Alanine Racemase--
A search for structural
alignment using DALI (33) revealed high similarity (Z-value 34.4) with
eukaryotic ODCs, enzymes found in the polyamine biosynthetic pathway
catalyzing the decarboxylation of ornithine to putrescine and a lower
level of structural similarity with AR from B. stearothermophilus (Z-value, 18.3). Multiple sequence alignments
of known mycobacterial DAPDC sequences, eukaryotic ODCs with known
structures, and B. stearothermopilus AR are presented in
Fig. 3 and summarized together with
structural data in Table III. Despite the
relatively low level of amino acid sequence identity between eukaryotic
ODCs and M. tuberculosis DAPDC (~18%), least squares
superposition of the structures indicates close resemblance (r.m.s.d.
values, ~2.2 Å). Even AR, which shares only 5% identity with DAPDC,
superimposes with 2.7 Å r.m.s.d. (Fig.
4). The higher deviation can be
attributed largely to a distinct rotation of the AR
-domain
respective to the well superimposing
/
barrels (~30°, see
also Grishin et al. 1999 (34)).

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Fig. 3.
Multiple sequence alignment of
PLP-dependent enzymes. Top line
indicates regions of partially conserved or important binding motives
or residues. Alignment carried out with ClustalW 1.8.2 (40). Color key:
green, polar residues; red, hydrophobic residues;
blue, negatively charged; and magenta, positively
charged.
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Fig. 4.
Backbone superposition of known fold
type III PLP-dependent enzyme structures. Panel
A, color key: cyan, M. tuberculosis DAPDC;
magenta, human ODC; green, mouse ODC; and
yellow, T. brucei. Panel B,
superposition of M. tuberculosis DAPDC (cyan)
with B. stearothermopilus AR (red). The
rotation of the AR -domain relative to the other structures is
clearly visible. The superpositions were carried by the
Local-Global-Alignment server (Adam Zemla,
predictioncenter.llnl.gov/local/lga/lga.html); corresponding
r.m.s.d. values are listed in Table III. The figure was prepared using
Swiss Pdb Viewer (41) and PovRay (www.povray.org).
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The sequence alignments (Fig. 3) also show a decreasing conservation of
PLP binding motives from the mycobacterial DAPDCs to the eukaryotic
ODCs and AR. The KAFL motif, containing the lysine residue that
covalently binds to PLP via Schiff base (internal aldimine) formation,
is conserved in procaryotic DAPDCs and eukaryotic ODCs, as is the
glycine rich motif (GGG) shown to interact with the phosphate group of
PLP. Other conserved motifs include the HIGS motif (thought to be
involved in protonation/deprotonation reactions), and the EPGR and
CESGD motifs, which are part of the substrate binding regions (35).
These motifs, however, with the exception of EPGR, appear not conserved
in the structurally related alanine racemase, consistent with its very
low sequence identity to the DAPDCs.
Comparison of the large, buried, solvent-accessible surface area at the
dimer interface (Table III) indicates that DAPDC (3462 Å2,
or 21%) forms the most stable dimer among the fold type III members of
known structure. The extensive number of conserved intermolecular
contacts and the absence of extended crystal packing contacts (largest
contact area between symmetry related subunits in the crystal lattice
is 64 Å2) indicate that DAPDC is an obligate dimer.
Additional structural support for the dimer as the functional unit
comes from the unexpected finding of a disulfide bridge between Cys-93
of one subunit of the dimer and Cys-375 of the other subunit.
Intersubunit disulfide bridges are very rare in cytoplasmic proteins,
especially in prokaryotes. Cys-93 is found only in mycobacterial DAPDCs
but is absent in all other bacterial DAPDCs. Cys-375 also forms a
hydrogen bond via its backbone oxygen to the PLP OP3 hydroxyl group of
the other subunit and is conserved in all bacterial DAPDCs as well as
in other type III B6-dependent enzymes.
Chromatographic experiments further provide chemical evidence that
M. tuberculosis DAPDC is indeed a stable dimer. DAPDC
migrated with an apparent molecular weight consistent with a dimer in
gel filtration chromatography experiments, and the disulfide bridge
adjoining the two subunits was confirmed by non-reducing SDS-PAGE (not
shown). Interestingly, early ultracentrifugation studies reported that
E. coli DAPDC was a tetramer (36), whereas gel filtration
analysis suggested that the E. coli DAPDC enzyme was
monomeric (37). For M. tuberculosis DAPDC, neither the
crystal structure nor size exclusion chromatography nor the native SDS
gels described above support a monomeric state or the formation of a tetramer.
The PLP-binding Site--
The active site of M. tuberculosis DAPDC is located in a shallow, highly
hydrophilic cavity between the dimer interfaces with the deep PLP
binding pocket located near the C-terminal ends of the
strands of
the
/
barrel, similar to other ODCs (14-16). Clear electron
density for PLP was visible in the SNW omit maps of the ternary complex
and indicated the presence of a covalent C=N link between Lys-72 N
and C4A of PLP (Fig. 5).

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Fig. 5.
Electron density in the DAPDC binding cleft
for covalently bound PLP and lysine. Both PLP and lysine were
omitted from the model before map generation (Shake&wARP map (31)
contoured at 1 level). The blob feature in XtalView has been used
to limit the display of the electron density within 2 Å of the
model. This figure was created by XtalView (28) and rendered with
Raster3d (42).
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Hydrogen bonds and nonbonding contacts between PLP and DAPDC are
summarized in Fig. 6. The oxygen atoms of
the PLP phosphate group hydrogen bond with the peptide backbone
nitrogen atoms of Gly-258 in the glycine rich motif and those of
Gly-302 and Arg-303. OP1 also forms a hydrogen bond with the hydroxyl
group of Tyr-405. In the DAPDC-lysine binary complex, a sulfate ion
occupies the same position as the phosphate group of PLP in the ternary
DAPDC-PLP-lysine structure.

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Fig. 6.
Schematic representation of ligand binding
interactions in active site pocket of DAPDC. Residues of both
homodimer subunits contribute to PLP and to lysine binding. This figure
was created by LIGPLOT (43).
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In addition to the covalent link to Lys-72 N
, the pyridyl moiety of
PLP is positioned by a hydrogen bond to the side-chain carboxylate of
Glu-300, which participates in an extended hydrogen bond network with
Asp-91 and the conserved residues Asp-254 and His-211. Two histidine
residues (114 and 213) and Ala-70 form hydrophobic contacts, with
His-213
-stacking against the si face of the pyridine
ring. His-114 and Asp-91 are positioned toward the re face
of the pyridine ring, and both are within hydrogen bonding distance of
the carboxylate of Glu-300. The network of interactions around Glu-300
in the binding pocket essentially fixes the position of the imidazole
side chains of His-114 and His-211, as well as the carboxylates of
Asp-91 and Asp-254 with respect to the pyridine ring of PLP. An
additional hydrogen bond to the other subunit of the dimer exists
between the O3 hydroxyl group of PLP(B) and the backbone oxygen of the
disulfide forming cysteine Cys-375A (Fig. 6).
The side chain of His-213
-stacks with the si side of the
pyridyl ring. This residue is conserved in the eukaryotic ODCs; however, His-211 and His-114 are absent and are replaced by serine and
alanine or glycine, respectively. In B. stearothermopilus AR, the
-stacked His-213 is again conserved via AR His-166, and His-211 and His-114 are replaced by Tyr-164 and Leu-85. The highly variable environment on the re face of the pyridyl ring
caused by these residue substitutions could play a significant role in fine-tuning the (stereo)specificity and/or pH optimum of the different PLP-mediated reactions in these enzymes.
Lysine Binding to M. tuberculosis DAPDC--
In the
DAPDC-PLP-lysine complex, the density for reaction product lysine could
be located in each binding site. In binding site B, the density is very
clear and allowed unambiguous positioning and refinement of the lysine
molecule (Fig. 5). In site A, the lysine is again oriented similarly to
the first site, but its exact position along the channel opening in the
binding site is not as clear as for site B. Both lysines are positioned
with the side chain toward the si face of the PLP pyridyl
ring, consistent with decarboxylation occurring on this side of the
ring. Residues of domain II of the other subunit (Ser-377A, Glu 376A)
participate in lysine binding consistent with the important role of ODC
Asp-361 (corresponding to DAPDC Glu-376) that has been demonstrated in Ala mutation studies (38), which show a 2000-fold decrease in substrate
binding affinity in mODC. The carboxyl group of lysine is further fixed
by conserved residue Arg-303, which participates in PLP binding via
backbone N contacts as well. As clearly visible in the electron density
Fig. 5, the
-amino group and CE of lysine are positioned reasonably
close (~4.0 Å) to the catalytic Schiff base formed by the Lys-PLP
internal aldimine. (Fig. 5). A model of the substrate DAP based on the
bound lysine would thus have its (D)-aminoacyl group in a
position to interact with the internal aldimine from the si
side of the pyridoxyl ring as well as with conserved His-213, Arg-161,
and possibly Ser-377.
Given the limited 2.6 Å resolution of the present structure, further
discussion of the details of the stereospecificity of the
decarboxylation mechanism in DAPDC must remain speculative. The
structural similarity between the DAPDC binding site and that of
eukaryotic ODCs suggests a related mechanism. The catalytic mechanism
of the decarboxylation reaction preformed by ODCs has been extensively
studied (14, 38). The major difference is that in ODCs the amino acid
substrate ornithine is in an L configuration, but DAPDC
decarboxylates the D-aminocarboxyl group of
meso-DAP. Details in the orientation of the
D-aminocarboxyl group with respect to the conjugated
pyridyl ring system acting as an electron sink as well as
stereospecificity of the anchoring of the non-reacting L-aminocarboxyl group through the domain II residues are
likely responsible for achieving stereospecific decarboxylation of DAP. Amino acid decarboxylation reactions of fold type III
PLP-dependent enzymes generally occur on the si
side of the pyridyl ring plane (as discussed by Kern et al.
in 1999 (15)), and evidence exists that the reaction may involve an
inversion of the reactive C
of the substrate (39).
Structural Basis of DAPDC as a Potential Anti-tuberculosis Drug
Target--
The comparison of DAPDC with the inhibitor- and
product-bound ODC structures (14, 32) of the parasitic flagellate
Trypanosoma brucei indicates that DAPDC, given that it is
essential for M. tuberculosis viability, could be a
potential anti-mycobacterial drug target. Although there are currently
no known drugs that target DAPDC, one of the most widely used drugs
used to treat African sleeping sickness is
-difluoromethylornithine
(DFMO), a suicide inhibitor that targets T. brucei ODC (32).
In the crystal structure, DFMO forms the external aldimine linkage with PLP as seen in the product-bound structure (14), but in addition it is
covalently bound to the side chain of Cys-360, thus irreversibly blocking the binding site (Fig. 5). A slight backbone torsion, combined
with an ~160° rotation of the equivalent Cys-375 SG, suffices to bring DAPDC into practically the same conformation as the
DFMO-bound T. brucei ODC (Fig.
7) but necessitates the breakage of the
intersubunit disulfide bond in DAPDC. It has been proposed that in ODCs
DFMO decarboxylation via the internal PLP aldimine followed by
elimination of a F
anion might form a highly reactive
electrophilic imine, attacking the nucleophilic Cys-360 thiol group
(35). To what degree a reactive imine of a fluorinated DAP analogue
might be capable of attacking the Cys-375-Cys-93 disulfide bond, is
unknown. It certainly would require a transient conformational
rearrangement, probably associated with a slight rotation of PLP, which
now has lost its covalent link, to position the reactive imine so that a reaction can take place. Provided the disulfide bond gets broken, the
product conformation would closely resemble the arrangement found in
DFMO-bound ODC. An energy-minimized model, starting from a DAP molecule
placed just as the bound DFMO in the T. brucei x-ray
structure, shows that the same conformation is conceivable for a
putative DAPDC-inhibitor complex, with quite satisfying geometry (Fig.
7). Stereospecificity of the decarboxylation reaction preceding the
attack of the reactive imine intermediate would likely require that a
DAP analog be stereospecifically fluorinated at the
D-aminocarboxyl group of DAP.

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Fig. 7.
Stereoviews of superpositions of the active
sites of the two models. A, superposition of
energy-minimized models of putative DAPDC-inhibitor (DFDAP) complex
(green carbon backbone) with ternary DAPDC-PLP-Lysine
complex (cyan carbon backbone) are shown in stereo.
The aminocarboxyl group on the PLP-bound DFDAP molecule occupies a
position similar to lysine in the ternary DAPDC complex. The
DAPDC-DFDAP was modeled covalently bound to Cys-375A, causing
speculation that breakage of the Cys-375A to Cys-93B intersubunit
disulfide bond could occur through an attack of a highly reactive
fluorinated imine intermediate (35). B, superposition of the
putative DAPDC-inhibitor (DFDAP) complex (green carbon
backbone) with the T. bruceii ODC-DFMO complex
(cyan carbon backbone), showing the similarity in the
overall geometry of the bound inhibitors in stereo. T. bruceii ODC-DFMO was superimposed onto the structure of DAPDC to
achieve a crude positioning of the PLP-DFMO complex in the active site
of the tuberculosis (TB) enzyme. The PLP-DFMO complex was
extended to the corresponding bound PLP-DFDAP analog, and the starting
position was adjusted. Hydrogens were added, and the docked model was
refined further with BioMedCaChe (v.6.0a1). Valence and hybridization
checks were enabled and improved hydrogen bond lengths and van der
Waals interactions. The structure of DAPDC with the bound PLP-DFDAP
analog complex was optimized using the Bio-MM2 molecular mechanics
engine in CaChe.
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