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Structural insights into the alanine racemase from Enterococcus faecalis
Biochimica et Biophysica Acta 1794 (2009) 1030–1040
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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a p a p
Structural insights into the alanine racemase from Enterococcus faecalis Amit Priyadarshi a,b,1, Eun Hye Lee b,1, Min Woo Sung b, Ki Hyun Nam b, Won Ho Lee b, Eunice EunKyeong Kim a,⁎, Kwang Yeon Hwang b,⁎ a b
Biomedical Research Center, Life Science Division, Korea Institute of Science and Technology, Seongbuk-gu, Seoul 136-791, South Korea Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-701, South Korea
a r t i c l e
i n f o
Article history: Received 3 December 2008 Received in revised form 19 February 2009 Accepted 4 March 2009 Available online 26 March 2009 Keywords: Alanine racemase D-cycloserine PEG PLP Enterococcus faecalis
a b s t r a c t Alanine racemase (AlaR) is a bacterial enzyme that belongs to the fold-type III group of pyridoxal 5′phosphate (PLP)-dependent enzymes. AlaR catalyzes the interconversion between L- and D-alanine, which is important for peptidoglycan biosynthesis. This enzyme is common in prokaryotes, but absent in eukaryotes, which makes it an attractive target for the design of new antibacterial drugs. Here, we report the crystal structures of both the apoenzyme and the D-cycloserine (DCS) complex of AlaR from the pathogenic bacterium Enterococcus faecalis v583, at a resolution of 2.5 Å. DCS is a suicide inhibitor of AlaR and, as such, serves as an antimicrobial agent and has been used to treat tuberculosis and urinary tract infection-related diseases, and makes several hydrogen bonds with the conserved active site residues, Tyr44 and Ser207, respectively. The apoenzyme crystal structure of AlaR consists of three monomers in the asymmetric unit, including a polyethylene glycol molecule in the dimer interface that surrounds one of the His 293 residues and also sits close to one side of the His 293 residue in the opposite monomer. Our results provide structural insights into AlaR that may be used for the development of new antibiotics targeting the alanine racemase in pathogenic bacteria. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Among the 20 species of the genus Enterococcus, Enterococcus faecalis (E. faecalis) is most commonly associated with human diseases. Gram-positive enterococci are naturally resistant to penicillin and cephalosporins, and can also become resistant to other drugs via the acquisition of foreign DNA [1]. E. faecalis is a leading cause of nosocomial UTIs (urinary tract infections) and may bind to bladder epithelial cells, setting the stage for secondary, more severe infections. Despite the current recognition of E. faecalis as an important uropathogen, much remains to be determined regarding the pathogenicity of nosocomial UTIs [2]. Bonten et al. reported that E. faecalis plays an etiological role in polymicrobial pneumonia, although a previous study indicated that this species occasionally caused lung infections [3]. E. faecalis colonization and infection have been assumed to originate from endogenous sources. However,
⁎ Corresponding authors. E.E. Kim is to be contacted at Korea Institute of Science and Technology, Seongbuk-gu, Seoul 136-791, South Korea, Tel.: +82 2 958 5937. K.Y. Hwang, Division of Biotechnology, College of Life Science and Biotechnology, Korea University, Seoul 136-701, South Korea, Tel.: +82 2 3290 3009; fax. +82 2 923 3225. E-mail addresses: [email protected] (E.E. Kim), [email protected] (K.Y. Hwang). 1 These authors contributed equally to this work. 1570-9639/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2009.03.006
previous studies have demonstrated that colonization of the upper respiratory tract by E. faecalis is frequently preceded by gastric colonization, thereby suggesting that colonization occurs via the gastro-pulmonary route [3]. Alanine racemase (AlaR, EC 5.1.1.1) catalyzes the interconversion of D- and L-alanine and plays an important role in supplying Dalanine in most bacterial strains. A peptidoglycan layer protects bacteria from osmotic lysis, and D-alanine is essential for cell wall peptidoglycan biosynthesis [4]. Cephalosporins disrupt the synthesis of the peptidoglycan layer of bacterial cell walls. The final transpeptidation step in the synthesis of the peptidoglycan is facilitated by transpeptidases known as penicillin-binding proteins (PBPs). PBPs bind to the D-Ala-D-Ala moiety at the end of muropeptides (peptidoglycan precursors), and crosslink the peptidoglycan. Beta-lactam antibiotics (such as penicillin) mimic this PBP-binding site and competitively inhibit PBP crosslinking of peptidoglycan. Defects in, or disruption of, the cell wall peptidoglycan is lethal to bacteria, and AlaR is therefore an important target for the design of new antibacterial drugs [5]. AlaR is a member of the fold-type III group of pyridoxal 5′phosphate (PLP) dependent enzymes [6]. AlaR is one of the enzymes for the peptidoglycan layer production in both Gram-negative and Gram-positive bacteria, and requires PLP as a cofactor. PLP binds covalently to the protein via an imine bond to the -amino group of a lysine side chain, forming an internal aldimine (Schiff's base) form [7]. All known bacteria require D-alanine for peptidoglycan biosynthesis,
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whereas only L-alanine is employed in the synthesis of eukaryotic proteins [8]. AlaR utilizes a “two-base” mechanism, employing two active site residues during catalysis. Two conserved residues of the active site, i.e., lysine and tyrosine, function as a catalytic acid and a base, generate the enantiomeric product D-alanine from L-alanine, thereby playing an important role in the aldimine to ketimine conversion [6,9,10]. Cycloserine (4-amino-3-isoxazolidinone) is a naturally occurring inhibitor of most PLP-dependent enzymes. Both L- and Denantiomers of cycloserine act as irreversible suicide inhibitors of AlaR [11,12] and can thus be used as antimicrobial agents. The Lisomers of amino acids are frequently found in nature and are generated by a variety of biosynthetic pathways. Presumably, AlaR-specific inhibitors would kill bacteria but would not have adverse side effects for humans, as no known mammalian AlaR homologs exist. The catalytic activity of AlaR depends on the binding of the PLP cofactor, a phosphorylated and oxidized form of vitamin B6. However, formation of the enantiomeric mates normally requires some form of chiral reduction, or an enzymatically-catalyzed equilibration about the R-carbon between the two isomers [12]. The AlaR enzyme occurs ubiquitously in eubacteria and is indispensable, because D-alanine is an essential component of the peptidoglycans in eubacterial cell walls. Therefore, the enzyme is an attractive target for the development of mechanism-based inactivators, which may prove useful as antibacterial agents [6]. In order to elucidate the structural and functional features of AlaR from E. faecalis (EfAlaR), we expressed, purified and crystallized both the apoenzyme and the D-cycloserine (DCS) complex and conducted an X-ray crystallographic analysis. The structural information acquired in this study will provide insights into the development of new antibiotics. 2. Materials and methods 2.1. Sequence alignment Multiple sequence alignment of AlaR isozymes was performed using CLUSTAL X 1.83 [13]. The amino acid sequences of the enzymes were obtained from the NCBI website (http://www.ncbi.nih.gov). Sources and protein accession numbers are as follows: E. faecalis, Q837J0; Geobacillus stearothermophilus, P10724; Haemophilus influenzae, P45257; Escherichia coli, P0A6B4; Pseudomonas aeruginosa, Q9HTQ2; Mycobacterium tuberculosis, P0A4X2; Helicobacter pylori, Q1XG01. 2.2. Protein purification, crystallization and data collection The alanine racemase gene (alr, 1116 bp; accession no. AE016830) was amplified by polymerase chain reaction (PCR) from the genomic DNA of E. faecalis as described elsewhere [14]. Protein purification and crystallization conditions were the same for the apoenzyme and the AlaR–DCS complex. The apoenzyme crystal was obtained using a buffer containing 0.1 M HEPES (pH 8.0), 22% (w/v) PEG 8000 and 0.3 M calcium acetate, with cyclohexyl-methyl-β-D-maltoside added to one-tenth of the total volume. For the DCS complex, the apoenzyme crystal was soaked in 5 mM D-cycloserine for 2 h prior to diffraction. Glycerol 30% (v/ v) was used as the cryoprotectant. X-ray diffraction data were collected from the cooled crystals using an ADSC Quantum CCD 210 detector at beamline 4A MXW at the Pohang Light Source (PLS), South Korea. X-ray diffraction data of EfAlaR at a resolution of 2.5 Å for the apoenzyme and DCS complex were collected at the PLS. All of the data were integrated and scaled using the HKL2000 program [15]. Data collection and refinement statistics are presented in Table 1.
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Table 1 Data collection and refinement statistics. Data collection
Apoenzyme
Complex
Space group Resolution (Å) a, b, c, (Å) No. unique observations Completeness (%) Rsyma I/σ (I) Refinement statistics Resolution (Å) Completeness (%) Rwork Rfree r.m.s.d. Length (Å) Angle (°) Average B-factor Ramachandran favorite (%) -Most favored region -Additionally allowed region -Generously allowed region
C2221 50–2.50 (2.59–2.50) 94.634, 156.516, 147.878 37447 (3584) 97.9 (95.3) 0.145 (0.331) 17.7 (3.02)
C2221 50–2.50 (2.59–2.50) 94.597, 156.186, 146.930 35277 (3209) 95.7 (88.4) 0.110 (0.259) 14.9 (2.84)
20–2.5 (2.66–2.50) 97.9 (94.2) 0.216 0.284
50–2.5 (2.59–2.50) 92.8 (87.2) 0.216 0.267
0.007 1.38 35.5 100 84.3 14.3 1.4
0.008 1.31 35.9 100 84.9 14.8 0.3
Numbers in parentheses are for the outer shell. a Rsym = ∑(I − bIN) / ∑bIN, where I is the intensity measurement for a given refraction and bIN is the average intensity for multiple measurements of this refraction.
2.3. Structure determination The crystal structure of EfAlaR was solved by molecular replacement using GsAlaR (PDB ID 1EPV) as a starting model. Three translated positions were found within the asymmetric unit using CNS [16]. Multiple cycles of editing and adjustment of the model into σ A-weighted 2Fo-Fc and Fo-Fc were performed using the Coot program [17]. Further simulated annealing, energy minimizations and individual isotropic B-factor refinement were carried out in CNS [16]. The final models were validated with PROCHECK [18]. The primary model of the EfAlaR was built based on the phase obtained from the molecular replacement solution with the GsAlaR structure, and the apoenzyme EfAlaR structure was subsequently used as a model for the DCS complex structure using CCP4 [19]. 3. Results 3.1. Sequence alignment The sequences of seven distinct isozymes of alanine racemase were aligned using the CLUSTAL X 1.83 algorithm, and the results are shown in Fig. 1. A strong sequence similarity was found between the alanine racemases from various organisms. Regions of highest homology were found near the N-terminus of the enzymes as well as in the sequences surrounding the active site residues (Lys40 and Tyr267 in E. faecalis). These key active site residues and their equivalent position in seven distinct AlaR are shown in Table 2. The alignment shows that the amino acid sequence identity between EfAlaR and AlaR from G. stearothermophilus is 49%, H. influenzae 32%, E. coli 30%, P. aeruginosa 34%, M. tuberculosis 36%, and H. pylori 34%. 3.2. Structure determination and refinement EfAlaR crystals of the apoenzyme and the DCS complex (1.2 mm × 0.2 mm × 0.15 mm in size) suitable for X-ray diffraction were obtained using optimized crystallization conditions, and diffracted to a resolution of 2.5 Å. The auto-indexing procedure performed with DENZO, scalepack using the HKL2000 suite, indicates that the crystals belong to an orthorhombic space group, with unitcell parameters of a = 94.634, b = 156.516, c = 147.878 Å, and α = β = γ = 90°. The DCS complex crystal has slightly different unit-
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Fig. 1. Multiple sequence alignment of seven isozymes of alanine racemase. Strictly conserved residues are indicated by (⁎); highly conserved residues are indicated by (:). The two catalytic residues are boxed. Sources and protein accession numbers are as follows: E. faecalis, Q837J0; G. stearothermophilus, P10724; P. aeruginosa, Q9HTQ2; H. influenzae, P45257; E. coli, P0A6B4; H. pylori, Q1XG01; and M. tuberculosis, P0A4X2.
cell parameters with a = 94.597, b = 156.186, c = 146.930. The space group was C2221 for both crystals, determined on the basis of systematic absences. The merged data set is 97.9% complete at a resolution of 2.5 Å for the apoenzyme and 95.7% for the DCS complex.
Assuming the presence of three molecules per asymmetric unit, the calculated Matthews coefficient (VM) value is 2.26 Å3 Da− 1 [20]. The solvent content of the crystal was calculated to be 45.5%. Data refinement statistics are presented in Table 1.
A. Priyadarshi et al. / Biochimica et Biophysica Acta 1794 (2009) 1030–1040 Table 2 Conserved catalytic residues of various AlaR enzymes and their equivalent position in seven pathogenically important AlaR enzymes. AlaR source
Catalytic residues
AlaR (E. faecalis) AlaR (G. stearothermophilus) AlaR (H. influenzae) AlaR (E. coli) AlaR (P. aeruginosa) AlaR (M. tuberculosis) AlaR (H. pylori)
Lys40 Lys39 Lys36 Lys34 Lys33 Lys42 Lys37
Tyr267 Tyr265 Tyr256 Tyr255 Tyr253 Tyr271 Tyr271
The EfAlaR structure was solved by molecular replacement with CNS [16], using a monomer of the G. stearothermophilus AlaR structure (PDB ID 1EPV). The final models of the apoenzyme and the DCS complex, validated with PROCHECK[18], show that 100% of the residues lying within the allowed regions of the Ramachandran plot. For the DCS-inhibited structure, PROCHECK determined that 84.9% of all the residues were in the most favored regions of the Ramachandran plot, 14.8% of the residues were in the additionally allowed regions, and 0.3% of the residues were in the generously allowed regions. For the apoenzyme structure, PROCHECK determined that 84.3% of all the residues were in the most favored region of the Ramachandran plot, 14.3% of the residues were in the additionally allowed regions, and 1.4% of the residues were in the generously allowed regions. No residues were found in the disallowed regions of either structure. 3.3. Overall structure of the alanine racemase apoenzyme from E. faecalis The EfAlaR monomer is composed of two domains, an Nterminal and a C-terminal domain. The N-terminal domain, consisting of residues 1–229, forms a TIM barrel structure, which provides space for the active site. The N-terminal domain of EfAlaR is composed of nine α-helices (α 1–9), eight β-strands (β2–9), a PLP cofactor, and acetate. The first β-strand of the monomer is located at the extreme N-terminus of the EfAlaR structure, and does not contribute to the formation of the TIM barrel. Residues 247–371 constitute the second domain (C-terminal) of EfAlaR, and are almost completely composed of β-strands. Residues Leu230– Ala246, form a loop that connects the N-terminal with the Cterminal domain (Fig. 2). The overall structure of EfAlaR reveals that it is a homodimer in which two monomers form a head-to-tail association, such that the C-terminal domain of one monomer interacts with the N-terminal TIM barrel of the opposite monomer (Fig. 3). The EfAlaR homodimer is comprised of two active sites that catalyzed by two conserved residues Lys40 and Tyr267′ (from the other monomer) respectively. In the EfAlaR structure, a PLP cofactor binds covalently via an imine bond to the -amino group of the side chain of each Lys40, forming an internal aldimine (Schiff's base) form. The EfAlaR dimer is formed by a few polar interactions and several hydrophobic interactions, which play an important role in structure stabilization. The asymmetrical unit of the crystal consists of three molecules of EfAlaR (Fig. 3), but only the AC dimer represents the active conformation. There are approximately 17% (2900 Å2) surface contact areas (mostly hydrophobic interactions) between the A and C subunit; 72 residues of subunit A interact with 69 residues of subunit C (Fig. 4). In this EfAlaR structure, hydrophobic interactions play an important role in maintaining the dimer structure. The A and B subunits are just symmetric molecules in the crystal, as there are no effective interactions between them that would contribute to enzymatic activity. The dimer interface is composed primarily of residues located on various loops that connect the α-helices with the β-strands of the N-terminal domain
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of the first monomer, as well as loops connecting the β-strands of the C-terminal domain of the second monomer. Three active site residues participate at the dimer interface, Arg139, His169 and Tyr267′ (from opposite monomer). Each monomer binds to one PLP and acetate molecule in the active site, which is located towards the center of the TIM barrel. One PEG and one HEPES molecule bind along the surface of the dimer interface, between the A and C subunits. PEG and HEPES were included during the crystallization of EfAlaR. Interestingly, several water molecules were present at the dimer interface, making hydrogen bonds with residues and with other water molecules. 3.4. Comparison of the overall folding of alanine racemases Since 1997, AlaR structures from ten species of bacteria have been deposited in the RCSB Protein Data Bank. To elucidate the important features of this enzyme, AlaR structures from three important pathogens, AlaR from G. stearothermophilus (PDB ID 1SFT) [21], DadX from P. aeruginosa (PDB ID 1RCQ) [22], and AlaR from M. tuberculosis (PDB ID 1XFC) [23], were compared with EfAlaR. When we superimposed these AlaR structures with EfAlaR, we established that the C-terminal domain is well conserved among the four structures, but the N-terminal domain shows some variability (Fig. 5). The r.m.s. deviations between EfAlaR and the Gram-positive G. stearothermophilus AlaR (GsAlaR) and M. tuberculosis AlaR (MtAlaR) are 0.920 and 1.346, respectively, whereas, the r.m.s. deviation between EfAlaR and the Gram-negative P. aeruginosa DadX (PaDadX) is 1.924. All the eubacteria mentioned above possess alanine racemases for peptidoglycan biosynthesis. However, some bacteria, such as P. aeruginosa, possess additional catabolic alanine racemases that are distinct from other AlaR [24]. The secondary structure and
Fig. 2. Monomer structure of EfAlaR. The N-terminal domain has a TIM barrel structure composed of nine α-helices (α 1–9) and eight β-strand (β2–9). The first βstrand of the monomer is located at the extreme end of the N-terminus of the protein and does not contribute to the TIM barrel. The C-terminal domain is mostly composed of β-strands. PLP (pyridoxal 5′-phosphate) and Ace (acetate) are located near the TIM barrel.
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Fig. 3. Overall structure of EfAlaR in an asymmetrical unit. Three subunits are shown in cyan, yellow and green. Subunits A and C form the active dimer. PLP (pyridoxal 5′-phosphate), Ace (acetate), PEG (polyethylene glycol), and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) are shown at the dimer interface.
overall folding of EfAlaR is very similar to that observed for GsAlaR, MtAlaR and PaDadX, confirming a strong architectural resemblance between these prokaryotic enzymes. The regions near the active site and dimer interface superimpose very well between the EfAlaR and GsAlaR structures. Minor structural
differences exist in areas that do not participate in dimerization. In contrast, only one domain of PaDadX can be superimposed on the EfAlaR structure. Whereas the N-terminal domain is well superimposed, but, the C-terminal domain of PaDadX is rotated 15° relative to that of EfAlaR (Fig. 5).
Fig. 4. Interacting residues (buried region) between subunits A and C of EfAlaR show mostly hydrophobic interactions. The electrostatic potential mapped on-to the molecular surface of the EfAlaR dimer interface is shown separately. The electrostatic potential was computed with PYMOL.
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Fig. 5. Superposition of various alanine racemases. AlaR from Enterococcus faecalis (EfAlaR) is shown in cyan, AlaR from Geobacillus stearothermophilus (PDB ID 1EPV) in pink, DadX from Pseudomonas aeruginosa (PDB ID 1RCQ) in yellow, and AlaR from Mycobacterium tuberculosis (PDB ID 1XFC) in green. The N-terminus shows structural variability between these four AlaR structures, and the C-terminus of PaDadX is tilted 15° relative to the EfAlaR axis.
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two different monomers (Fig. 6b), i.e., residues Asp174, Glu175, Ile176, Asn231, Ser233, Lys236, Ile354 and His355 from the first monomer, and residues Tyr267′, Tyr286′, Arg292′ and Arg311′ from the second partner monomer. Among these residues, Asp174, Pro232, Tyr356, Tyr267′, Tyr286′, Arg292′ and Arg311′ are highly conserved across AlaR (Fig. 1). By investigating the charges of the residues around the entryway, we discovered that the two residues with negative charges, namely Asp174, Glu175, are positioned almost opposite the Tyr267′ residue from the partner monomer (Fig. 6b). The electrostatic surface charges around the substrate entryways of EfAlaR, and its homologs, PaDadX, GsAlaR and MtAlaR, were found to be similar. The substrate entry corridor is about ∼10.0 Å long and leads towards the reactive atom C4′of PLP. In the EfAlaR structure, the opening width of the active site corridor between Tyr356 and Tyr267′ is 6.4 Å, wider than that between the corresponding tyrosine residues in the active site of PaDadX [22] (i.e. 2.7 Å), and this increases accessibility to the substrate (van der Waals radius of 3.4 Å). Asp174, which is fully conserved across AlaR homologs (Fig. 1), forms a middle gate with Arg292′, whereas the inner gate is formed by Tyr267′ and Tyr356 near the active site pocket. Alanine racemases use several types of interactions to stabilize the substrate carboxylate group. In EfAlaR, the side chains of Arg139, and to some extent of Tyr267′, and in GsAlaR the main chain of Met 314′ [29] and Tyr354 [30], play important roles in stabilization. A conserved residue, Arg222, is unprotonated throughout the catalytic cycle because of its interaction with pyridine N. A point mutant of this residue, R222E, exhibits a decrease in kcat values of up to three fold [6], revealing that this residue is also important for stabilizing AlaR.
3.5. Active site and substrate entryway
3.6. PEG and HEPES molecules
The geometry of the active site's binding pocket (6.0 × 4.0 × 3.0 Å) of EfAlaR and of other reported AlaR structures are quite similar. In the EfAlaR structure, two residues function as the catalytic acid and base, with the Lys40 residue attacking the α-hydrogen of L-alanine to form a carbanionic intermediate, and the Tyr267′ donating a proton from the opposite side, generating the enantiomeric product, consistent with previous reports [25,26]. The conserved active site of EfAlaR, composed of these two residues along with a hydrogen-bonded network of residues, is shown in Fig. 6a. This hydrogen-bonded network plays an important role in the reaction mechanism, as noted in the description of previous AlaR structures [10,12,21,22,23]. The active site is solvent-accessible, one water molecule forms H-bonds with the oxygen atom of PLP (2.8 Å) and two additional H-bonds with the oxygen atoms from the peptide backbone or side chains of His169 and Ala208. The alanine racemization reaction is initiated via Schiff base formation between the substrate and cofactor (PLP). The substrate Rcarbon is then deprotonated and stabilized by electron delocalization via the electrophilic pyridinium nitrogen. We observed that each PLP is near the center of the TIM barrel and closer to the C-terminal domain of the partner monomer. In the EfAlaR structure, PLP concomitantly interacts with the Tyr44, Arg139, Arg222 and Tyr356 residues (Fig. 6a). The conjugated π system of the PLP ring is the functional group that reacts with the α-carbon of the substrate and plays a pivotal role in AlaR activity [27,28]. We found that the conserved Arg139 and Met314′ residues are positioned such that they are capable of forming hydrogen bonds with the carbonyl oxygen of PLP, suggesting that the NH1 of Arg139 and the main chain nitrogen of Met314′ form the recognition site for the carboxyl group of the substrate and help to stabilize AlaR. In the EfAlaR structure, the residues responsible for instantly shaping the substrate entryway are represented by residues on the
The overall structure of EfAlaR is similar to that of GsAlaR, with the exception that EfAlaR has some additional electron density on the dimer interface. After repeated refinement, one PEG molecule and one HEPES molecule were revealed in the EfAlaR structure (Fig. 7a). The PEG molecule is located on the interface of the dimer, near the His293 and His293′ residues. The PEG molecule surrounds the His293 residue and lies partly between the two His residues (His293 and His293′), whereas the HEPES molecule is located on the opposite side of His293 (Fig. 7b). Based on the primary sequence alignment (Fig. 1), these two histidine residues in EfAlaR are replaced by arginine residues in the GsAlaR. The distance between PEG and its surrounding residues, His293 (3.8 Å), Lys350 (3.9 Å), Glu352 (3.5 Å), His293′ (3.1 Å), Arg311′ (3.90 Å), and Lys359′ (3.50 Å) reveals the hydrophilic nature of the compound (Fig. 7c). PEG was found to bind to the interface site, and not the catalytic residues, but they are positioned between the two active sites and the substrate entry site with approx. distance of 4.6 Å from entryway. Thus, this molecule could affect the catalytic activity of EfAlaR. Given the potential location and properties of this PEG binding pocket, this region could be used for designing inhibitors to gain better selectivity in the future. 3.7. Structure of the EfAlaR–DCS complex We determined the crystal structure of the DCS complex to 2.5 Å resolution. The presence of DCS in the active site does not induce any significant conformational changes in the enzyme. The r.m.s. deviation between the apoenzyme structure and the DCS complex structure is 0.32 Å. Our EfAlaR structure shows that the DCS is held in the active site by a network of hydrogen bonds, which are quite similar to those found in all the structures reported for this enzyme [10,12]. A
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Fig. 6. Active site residues and the substrate entry channel of EfAlaR. (a) Hydrogen bond network of the EfAlaR active site. Residues marked with (') belong to the partner monomer. Hydrogen bonds with an interatomic distance of less than 3.0 Å are depicted by dotted lines. (b) Electrostatic surface map around the substrate entry channel, residues from the two different monomers are shown in cyan and yellow, with PEG lying between two His293 residues.
carboxylate-binding motif has been observed interacting with the hydroxy-imine that forms part of the hydroxy-isoxazole ring. The hydroxyl moiety of DCS interacts with Tyr44, Arg139, Ser207, Arg222, and Tyr356 (Fig. 8a). The residues near DCS make a hydrophilic groove that enables them to form a hydrogen bond, which helps to stabilize EfAlaR (Fig. 8b). The electron density of the conserved Tyr267, which is approximately 3.0 Å distant from DCS, was found ordered in the EfAlaR–DCS complex structure, but was disordered in the GsAlaR structure (Tyr265 in GsAlaR) reported by Fenn et al. [12]. The PLP derivatives bound to cycloserine enantiomers may be identical, as observed in GsAlaR. About eleven residues were found to interact with DCS; among them, highly conserved residues, such as Tyr44, Ser207, Arg222 and Tyr356, were
less than 3.0 Å away from DCS (Table 3). One water molecule interacts with DCS. 3.8. Structural comparisons between the EfAlaR apoenzyme and the EfAlaR–DCS complex When we superposed the apoenzyme EfAlaR structure with that of the EfAlaR–DCS complex structure, no significant conformational changes were detected (Fig. 9). The spatial positions of the amino acid side chains near PLP and DCS are almost identical (Fig. 9). However, some corresponding regions of PLP and DCS cannot be superimposed due to conformational differences between the N1 of PLP and DCS. Active site residues were found in the same positions in
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Fig. 7. Ribbon diagram of the EfAlaR dimer. (a) PEG and HEPES molecules bound to EfAlaR. (b) Residues near PEG. Residues marked with (′) belongs to the partner monomer. (c) Electronegativity map of EfAlaR. PLP, pyridoxal 5′-phosphate; Ace, acetate; PEG, polyethylene glycol; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.
the apoenzyme and DCS complex structure; however, slight conformational changes occurred in the His169 and Arg222 side chains of subunit A and in the Tyr267′ and Met314′ side chains of subunit C
(Fig. 9). In the DCS complex structure, Arg139 from subunit A moves approximately 1.5 Å towards the DCS molecule as compared to the apoenzyme EfAlaR structure (Fig. 9). The average temperature factor
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Fig. 8. Residues of EfAlaR interacting with the D-cyloserine inhibitor. (a) Diagrammatic view of σA-weighted simulated annealing omit maps of DCS. Maps are shown at 1.5σ for the DCS adduct. This figure was generated with CNS xtal and CCP4 mapcover. (b) Electrostatic surface map around DCS.
for all atoms in both structures is approximately 35 Å2; however, almost double B-factor values were observed for the loop region Gln117–Asn124 in the DCS complex (Gln117–Arg128 in apoenzyme EfAlaR). Whereas, high B-factor regions, Pro145–Gln156 and Glu274– Glu277 of the apoenzyme are absent in EfAlaR–DCS complex structure. In the apoenzyme EfAlaR structure, an extra α-helix (α12) in subunit C and an extended β-strand (β4) in subunit A were observed (Fig. 9) that were not present in the EfAlaR–DCS complex. 4. Discussion AlaR is an attractive target for antibacterial drugs because of its absence in humans and its indispensable function in bacteria.
In retrospect of drug design many substrate analogues and inhibitors were discovered for the AlaR. Two natural antibiotics (D-cycloserine and O-carbamoyl-D-serine) and several alanine analogues (β,β,β-trifluoroalanine, alanine phosphonate, 1-aminocyclopropane phosphonate, and β- chloro- and β-fluoroalanine) are commonly used to inhibit AlaR. Though a combination of Xray crystallographic and spectroscopic methods was used to decipher the mechanisms of inhibition, it was found that these inhibitors have a fatal drawback, generating secondary-effect. They act not only on AlaR but also on all PLP-dependent enzymes, several of which are expressed in humans. Therefore, a detailed structural analysis of AlaR is needed in order to develop drugs with greater specificity.
A. Priyadarshi et al. / Biochimica et Biophysica Acta 1794 (2009) 1030–1040 Table 3 D-cycloserine interactions with the peptide backbone or side chains of various residues (polar interactions for hydrogen bonding and salt bridges). Residue (side chain)
D-cycloserine
Distance (Å)
VAL38 (CG1) LYS40 (CE) TYR44 (OH) LEU86 (CD1) ARG139 (NH1) HIS169 (ND1) ASN206 (CB) SER207 (OG) ARG222 (NE) VAL225 (CG2) TYR267 (OH) TYR356 (OH)
DCS DCS DCS DCS DCS DCS DCS DCS DCS DCS DCS DCS
3.59 3.63 2.56 3.73 3.12 3.48 3.59 2.50 2.97 3.72 3.06 2.60
(C5A) (C4A) (O2P) (C2) (O3) (C2) (O1P) (O1P) (N1) (O2P) (N) (O3P)
The structure of E. faecalis alanine racemase was solved by Xray crystallography for the apoenzyme and the DCS complex. The structure shows that the peptide backbone (Cα) positions of the apoenzyme and the DCS complex are quite similar. The conserved residue Tyr267 is constrained in the EfAlaR–DCS complex structure, which was found, more flexible in the GsAlaR structure. The PLP derivatives bound to cycloserine enantiomers were noticed with the same orientation as observed in the GsAlaR structure. From EfAlaR structures, we identified a different site that may be suitable for drug targeting, i.e., the PEG binding site. PEG was bound to His293 and His293′ along the dimer interface of AlaR, where the head of the PEG molecule was oriented approximately 4.6 Å away from the substrate entry pathway. This binding may affect the structural stability and activity of AlaR. To determine the affinity of PEG for EfAlaR we performed an isothermal calorimetry (ITC) experiment. However, repeated attempts of this experiment at various protein and PEG concentrations showed only very weak or no binding. These preliminary results will need to be confirmed by further experiments (using BIACORE or another technique). The substrate entryway is a key feature of AlaR, and has been proposed
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to be a potential target for drug design, since it consists of highly conserved residues. We predict that the negatively charged residues, Asp174 and Glu175, around the substrate entryway, play an important role in substrate orientation along with the cooperation of the positively charged Arg292′ and Arg311′ from the partner monomer. 5. Conclusion The EfAlaR structure is very similar to the alanine racemase structures reported from G. stearothermophilus, M. tuberculosis, and P. aeruginosa. While the hinge angles between the monomer domains of all four structures are uniquely different, but key active site residues superimpose well. Analysis of the EfAlaR structure confirms that this enzyme remains a challenging but important target for drug design. The most significant result of this structural report involves the entryway that leads to the active site. Our analysis reveals that the inner and middle regions of the protein are strongly conserved across species, while regions near the enzyme surface are more variable. AlaR has often been proposed to be a candidate target for designing antimicrobial drugs, given its essential role in prokaryotes but absence from eukaryotes. We can design AlaR inhibitors using various approaches, such as placing an inhibitor within the active site or blocking accessibility to the active site using an inhibitor that binds to the substrate entryway, or by designing inhibitors that would prevent dimerization. Based on the study of E. faecalis, which possesses resistance to cephalosporins and penicillin, the crystal structure of EfAlaR will aid in structurebased drug design. Acknowledgments We thank Dr. H. S. Lee and his staff for assistance during data collection at beamline 4A of Pohang Light Source, Korea. A. Priyadarshi and E. E Kim were supported by KIST grants. This study was supported by the Functional Proteomics Center, 21C Frontier Program of the Korea Ministry of Science and Technology.
Fig. 9. Superposition of D-cycloserine and PLP. D-cycloserine is shown in yellow and PLP in green. The enlarged β4 motif and the additional α12 observed in the apoenzyme EfAlaR structure are shown in green, and the EfAlaR–DCS complex is shown in cyan.
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References [1] A. Umeda, F. Garnier, P. Courvalin, M. Galimand, Association between the vanB2 glycopeptide resistance operon and Tn1549 in enterococci from France, J. Antimicrob. Chemother. 50 (2002) 253–256. [2] N. Shankar, C.V. Lockatell, A.S. Baghdayan, C. Drachenberg, M.S. Gilmore, D.E. Johnson, Role of Enterococcus faecalis surface protein Esp in the pathogenesis of ascending urinary tract infection, Infect. Immune 69 (2001) 4366–4372. [3] M.J.M. Bonten, C.A. Gaillard, F.H.V. Tiel, S.V. Geest, E.E. Stobberingh, Colonization and infection with Enterococcus faecalis in intensive care units: the role of antimicrobial agents, Antimicrob. Agents Chemother. 39 (1995) 2783–2786. [4] C.T. Walsh, Enzymes in the D-alanine branch of bacterial cell wall peptidoglycan assembly, J. Biol. Chem. 264 (1989) 2393–2396. [5] T.D.H. Bugg, C.T. Walsh, Intracellular steps of bacterial cell wall peptidoglycan biosynthesis: enzymology, antibiotics, and antibiotic resistance, Nat. Prod. Rep. 9 (1993) 199–215. [6] A. Watanabe, T. Yoshimura, B. Mikami, H. Hayashi, H. Kagamiyama, N. Esaki, Reaction mechanism of alanine racemase from Bacillus stearothermophilus: X-ray crystallographic studies of the enzyme bound with N-(5′-phosphopyridoxyl) alanine, J. Biol. Chem. 277 (2002) 19166–19172. [7] G.I. Mustata, T.A. Soares, J.M. Briggs, Molecular dynamics studies of alanine racemase: a structural model for drug design, Biopolymers 70 (2003) 186–200. [8] G. Mustata, J.M. Briggs, Cluster analysis of water molecules in alanine racemase and their putative structural role, Protein Eng. Des. Sel. 17 (2004) 223–234. [9] A. Watanabe, Y. Kurokawa, T. Yoshimura, T. Kurihara, K. Soda, N. Esaki, Role of lysine 39 of alanine racemase from Bacillus stearothermophilus that binds pyridoxal 5′-phosphate, J. Biol. Chem. 274 (1999) 4189–4194. [10] T.D. Fenn, T. Holyoak, G.F. Stamper, D. Ringe, Effect of a Y265F mutant on the transamination-based cycloserine inactivation of alanine racemase, Biochemistry 44 (2005) 5317–5327. [11] M.P. Lambert, F.C. Neuhaus, Mechanism of D-cycloserine action: alanine racemase from Escherichis coli, J. Bacteriol. 110 (1972) 978–987. [12] T.D. Fenn, G.F. Stamper, A.A. Morollo, D. Ringe, A side reaction of alanine racemase: transamination of cycloserine, Biochemistry 42 (2003) 5775–5783. [13] J.D. Thompson, T.J. Gibson, F. Plewniak, F. Jeanmougin, D.G. Higgins, The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools, Nucleic Acids Res. 25 (1997) 4876–4882. [14] A. Priyadarshi, E.H. Lee, M.W. Sung, J.H. Kim, M.-J. Ku, E.E. Kim, K.Y. Hwang, Overexpression, crystallization, and preliminary X-ray crystallographic analysis of the alanine racemase from Enterococcus faecalis v583, J. Microb. Biotech. 18 (2008) 55–58.
[15] Z. Otwinowski, W. Minor, Processing of X-ray diffraction data collected in oscillation mode, Methods Enzymol. 276 (1997) 307–326. [16] A.T. Brunger, P.D. Adams, G.M. Clore, W.L. DeLano, P. Gros, R.W. Grosse-Kunstleve, J.S. Jiang, J. Kuszewski, M. Nilges, N.S. Pannu, R.J. Read, L.M. Rice, T. Simonson, G.L. Warren, Crystallography and NMR system: a new software suite for macromolecular structure determination, Acta Crystallogr. D 54 (1998) 905–921. [17] P. Emsley, K. Cowtan, Coot: model-building tools for molecular graphics, Acta Crystallogr. D 60 (2004) 2126–2132. [18] P.A. Laskowski, M.W. McArthur, D.S. Moss, J.M. Thornton, PROCHECK: a program to check the stereochemical quality of protein structures, J. Appl. Crystallogr. 26 (1993) 283–291. [19] Collaborative Computational Project, Number 4, the CCP4 suite: programs for protein crystallography, Acta Crystallogr. D 50 (1994) 760–763. [20] B.W. Matthews, Solvent content of protein crystals, J. Mol. Biol. 33 (1968) 491–497. [21] J.P. Shaw, G.A. Petsko, D. Ringe, Determination of the structure of alanine racemase from Bacillus stearothermophilus at 1.9-Å resolution, Biochemistry 36 (1997) 1329–1342. [22] P. LeMagueres, H. Im, A. Dvorak, U. Strych, M. Benedik, K.L. Krause, Crystal structure at 1.45 Å resolution of alanine racemase from a pathogenic bacterium, Pseudomonas aeruginosa, contains both internal and external aldimine forms, Biochemistry 42 (2003) 14752–14761. [23] P. LeMagueres, H. Im, J. Ebalunode, U. Strych, M.J. Benedik, J.M. Briggs, H. Kohn, K.L. Krause, The 1.9 Å crystal structure of alanine racemase from Mycobacterium tuberculosis contains a conserved entryway into the active site, Biochemistry 44 (2005) 1471–1481. [24] J. Ju, H. Misono, K. Ohnishi, Directed evolution of bacterial alanine racemases with higher expression level, J. Biosci. Bioeng. 100 (2005) 246–254. [25] Y. Kurokawa, A. Watanabe, T. Yoshimura, N. Esaki, K. Soda, Transamination as a side-reaction catalyzed by alanine racemase of Bacillus stearothermophilus, J. Biochem. 124 (1998) 1163–1169. [26] M.J. Ondrechen, J.M. Briggs, J.A. McCammon, A model for enzyme–substrate interaction in alanine racemase, J. Am. Chem. Soc. 123 (2001) 2830–2834. [27] H.C. Dunathan, J.G. Voet, Stereochemical evidence for the evolution of pyridoxalphosphate enzymes of various function from a common ancestor, Proc. Natl. Acad. Sci. U. S. A. 71 (1974) 3888–3891. [28] C.G.F. Stamper, A.A. Morollo, D. Ringe, Reaction of alanine racemase with Laminoethylphosphonic acid forms a stable external aldimine, Biochemistry 37 (1998) 10438–10445. [29] A.A. Morollo, G.A. Petsko, D. Ringe, Structure of Michaelis complex analogue: propionate binds in the substrate carboxylate site of alanine racemase, Biochemistry 38 (1999) 3293–3301. [30] W.M. Patrick, J. Weisner, J.M. Blackburn, Site-directed mutagenesis of Tyr354 in Bacillus stearothermophilus alanine racemase identifies a role in controlling role in the evolution of antibiotic resistance, ChemBioChem 3 (2002) 789–792.
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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a p a p
Structural insights into the alanine racemase from Enterococcus faecalis Amit Priyadarshi a,b,1, Eun Hye Lee b,1, Min Woo Sung b, Ki Hyun Nam b, Won Ho Lee b, Eunice EunKyeong Kim a,⁎, Kwang Yeon Hwang b,⁎ a b
Biomedical Research Center, Life Science Division, Korea Institute of Science and Technology, Seongbuk-gu, Seoul 136-791, South Korea Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-701, South Korea
a r t i c l e
i n f o
Article history: Received 3 December 2008 Received in revised form 19 February 2009 Accepted 4 March 2009 Available online 26 March 2009 Keywords: Alanine racemase D-cycloserine PEG PLP Enterococcus faecalis
a b s t r a c t Alanine racemase (AlaR) is a bacterial enzyme that belongs to the fold-type III group of pyridoxal 5′phosphate (PLP)-dependent enzymes. AlaR catalyzes the interconversion between L- and D-alanine, which is important for peptidoglycan biosynthesis. This enzyme is common in prokaryotes, but absent in eukaryotes, which makes it an attractive target for the design of new antibacterial drugs. Here, we report the crystal structures of both the apoenzyme and the D-cycloserine (DCS) complex of AlaR from the pathogenic bacterium Enterococcus faecalis v583, at a resolution of 2.5 Å. DCS is a suicide inhibitor of AlaR and, as such, serves as an antimicrobial agent and has been used to treat tuberculosis and urinary tract infection-related diseases, and makes several hydrogen bonds with the conserved active site residues, Tyr44 and Ser207, respectively. The apoenzyme crystal structure of AlaR consists of three monomers in the asymmetric unit, including a polyethylene glycol molecule in the dimer interface that surrounds one of the His 293 residues and also sits close to one side of the His 293 residue in the opposite monomer. Our results provide structural insights into AlaR that may be used for the development of new antibiotics targeting the alanine racemase in pathogenic bacteria. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Among the 20 species of the genus Enterococcus, Enterococcus faecalis (E. faecalis) is most commonly associated with human diseases. Gram-positive enterococci are naturally resistant to penicillin and cephalosporins, and can also become resistant to other drugs via the acquisition of foreign DNA [1]. E. faecalis is a leading cause of nosocomial UTIs (urinary tract infections) and may bind to bladder epithelial cells, setting the stage for secondary, more severe infections. Despite the current recognition of E. faecalis as an important uropathogen, much remains to be determined regarding the pathogenicity of nosocomial UTIs [2]. Bonten et al. reported that E. faecalis plays an etiological role in polymicrobial pneumonia, although a previous study indicated that this species occasionally caused lung infections [3]. E. faecalis colonization and infection have been assumed to originate from endogenous sources. However,
⁎ Corresponding authors. E.E. Kim is to be contacted at Korea Institute of Science and Technology, Seongbuk-gu, Seoul 136-791, South Korea, Tel.: +82 2 958 5937. K.Y. Hwang, Division of Biotechnology, College of Life Science and Biotechnology, Korea University, Seoul 136-701, South Korea, Tel.: +82 2 3290 3009; fax. +82 2 923 3225. E-mail addresses: [email protected] (E.E. Kim), [email protected] (K.Y. Hwang). 1 These authors contributed equally to this work. 1570-9639/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2009.03.006
previous studies have demonstrated that colonization of the upper respiratory tract by E. faecalis is frequently preceded by gastric colonization, thereby suggesting that colonization occurs via the gastro-pulmonary route [3]. Alanine racemase (AlaR, EC 5.1.1.1) catalyzes the interconversion of D- and L-alanine and plays an important role in supplying Dalanine in most bacterial strains. A peptidoglycan layer protects bacteria from osmotic lysis, and D-alanine is essential for cell wall peptidoglycan biosynthesis [4]. Cephalosporins disrupt the synthesis of the peptidoglycan layer of bacterial cell walls. The final transpeptidation step in the synthesis of the peptidoglycan is facilitated by transpeptidases known as penicillin-binding proteins (PBPs). PBPs bind to the D-Ala-D-Ala moiety at the end of muropeptides (peptidoglycan precursors), and crosslink the peptidoglycan. Beta-lactam antibiotics (such as penicillin) mimic this PBP-binding site and competitively inhibit PBP crosslinking of peptidoglycan. Defects in, or disruption of, the cell wall peptidoglycan is lethal to bacteria, and AlaR is therefore an important target for the design of new antibacterial drugs [5]. AlaR is a member of the fold-type III group of pyridoxal 5′phosphate (PLP) dependent enzymes [6]. AlaR is one of the enzymes for the peptidoglycan layer production in both Gram-negative and Gram-positive bacteria, and requires PLP as a cofactor. PLP binds covalently to the protein via an imine bond to the -amino group of a lysine side chain, forming an internal aldimine (Schiff's base) form [7]. All known bacteria require D-alanine for peptidoglycan biosynthesis,
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whereas only L-alanine is employed in the synthesis of eukaryotic proteins [8]. AlaR utilizes a “two-base” mechanism, employing two active site residues during catalysis. Two conserved residues of the active site, i.e., lysine and tyrosine, function as a catalytic acid and a base, generate the enantiomeric product D-alanine from L-alanine, thereby playing an important role in the aldimine to ketimine conversion [6,9,10]. Cycloserine (4-amino-3-isoxazolidinone) is a naturally occurring inhibitor of most PLP-dependent enzymes. Both L- and Denantiomers of cycloserine act as irreversible suicide inhibitors of AlaR [11,12] and can thus be used as antimicrobial agents. The Lisomers of amino acids are frequently found in nature and are generated by a variety of biosynthetic pathways. Presumably, AlaR-specific inhibitors would kill bacteria but would not have adverse side effects for humans, as no known mammalian AlaR homologs exist. The catalytic activity of AlaR depends on the binding of the PLP cofactor, a phosphorylated and oxidized form of vitamin B6. However, formation of the enantiomeric mates normally requires some form of chiral reduction, or an enzymatically-catalyzed equilibration about the R-carbon between the two isomers [12]. The AlaR enzyme occurs ubiquitously in eubacteria and is indispensable, because D-alanine is an essential component of the peptidoglycans in eubacterial cell walls. Therefore, the enzyme is an attractive target for the development of mechanism-based inactivators, which may prove useful as antibacterial agents [6]. In order to elucidate the structural and functional features of AlaR from E. faecalis (EfAlaR), we expressed, purified and crystallized both the apoenzyme and the D-cycloserine (DCS) complex and conducted an X-ray crystallographic analysis. The structural information acquired in this study will provide insights into the development of new antibiotics. 2. Materials and methods 2.1. Sequence alignment Multiple sequence alignment of AlaR isozymes was performed using CLUSTAL X 1.83 [13]. The amino acid sequences of the enzymes were obtained from the NCBI website (http://www.ncbi.nih.gov). Sources and protein accession numbers are as follows: E. faecalis, Q837J0; Geobacillus stearothermophilus, P10724; Haemophilus influenzae, P45257; Escherichia coli, P0A6B4; Pseudomonas aeruginosa, Q9HTQ2; Mycobacterium tuberculosis, P0A4X2; Helicobacter pylori, Q1XG01. 2.2. Protein purification, crystallization and data collection The alanine racemase gene (alr, 1116 bp; accession no. AE016830) was amplified by polymerase chain reaction (PCR) from the genomic DNA of E. faecalis as described elsewhere [14]. Protein purification and crystallization conditions were the same for the apoenzyme and the AlaR–DCS complex. The apoenzyme crystal was obtained using a buffer containing 0.1 M HEPES (pH 8.0), 22% (w/v) PEG 8000 and 0.3 M calcium acetate, with cyclohexyl-methyl-β-D-maltoside added to one-tenth of the total volume. For the DCS complex, the apoenzyme crystal was soaked in 5 mM D-cycloserine for 2 h prior to diffraction. Glycerol 30% (v/ v) was used as the cryoprotectant. X-ray diffraction data were collected from the cooled crystals using an ADSC Quantum CCD 210 detector at beamline 4A MXW at the Pohang Light Source (PLS), South Korea. X-ray diffraction data of EfAlaR at a resolution of 2.5 Å for the apoenzyme and DCS complex were collected at the PLS. All of the data were integrated and scaled using the HKL2000 program [15]. Data collection and refinement statistics are presented in Table 1.
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Table 1 Data collection and refinement statistics. Data collection
Apoenzyme
Complex
Space group Resolution (Å) a, b, c, (Å) No. unique observations Completeness (%) Rsyma I/σ (I) Refinement statistics Resolution (Å) Completeness (%) Rwork Rfree r.m.s.d. Length (Å) Angle (°) Average B-factor Ramachandran favorite (%) -Most favored region -Additionally allowed region -Generously allowed region
C2221 50–2.50 (2.59–2.50) 94.634, 156.516, 147.878 37447 (3584) 97.9 (95.3) 0.145 (0.331) 17.7 (3.02)
C2221 50–2.50 (2.59–2.50) 94.597, 156.186, 146.930 35277 (3209) 95.7 (88.4) 0.110 (0.259) 14.9 (2.84)
20–2.5 (2.66–2.50) 97.9 (94.2) 0.216 0.284
50–2.5 (2.59–2.50) 92.8 (87.2) 0.216 0.267
0.007 1.38 35.5 100 84.3 14.3 1.4
0.008 1.31 35.9 100 84.9 14.8 0.3
Numbers in parentheses are for the outer shell. a Rsym = ∑(I − bIN) / ∑bIN, where I is the intensity measurement for a given refraction and bIN is the average intensity for multiple measurements of this refraction.
2.3. Structure determination The crystal structure of EfAlaR was solved by molecular replacement using GsAlaR (PDB ID 1EPV) as a starting model. Three translated positions were found within the asymmetric unit using CNS [16]. Multiple cycles of editing and adjustment of the model into σ A-weighted 2Fo-Fc and Fo-Fc were performed using the Coot program [17]. Further simulated annealing, energy minimizations and individual isotropic B-factor refinement were carried out in CNS [16]. The final models were validated with PROCHECK [18]. The primary model of the EfAlaR was built based on the phase obtained from the molecular replacement solution with the GsAlaR structure, and the apoenzyme EfAlaR structure was subsequently used as a model for the DCS complex structure using CCP4 [19]. 3. Results 3.1. Sequence alignment The sequences of seven distinct isozymes of alanine racemase were aligned using the CLUSTAL X 1.83 algorithm, and the results are shown in Fig. 1. A strong sequence similarity was found between the alanine racemases from various organisms. Regions of highest homology were found near the N-terminus of the enzymes as well as in the sequences surrounding the active site residues (Lys40 and Tyr267 in E. faecalis). These key active site residues and their equivalent position in seven distinct AlaR are shown in Table 2. The alignment shows that the amino acid sequence identity between EfAlaR and AlaR from G. stearothermophilus is 49%, H. influenzae 32%, E. coli 30%, P. aeruginosa 34%, M. tuberculosis 36%, and H. pylori 34%. 3.2. Structure determination and refinement EfAlaR crystals of the apoenzyme and the DCS complex (1.2 mm × 0.2 mm × 0.15 mm in size) suitable for X-ray diffraction were obtained using optimized crystallization conditions, and diffracted to a resolution of 2.5 Å. The auto-indexing procedure performed with DENZO, scalepack using the HKL2000 suite, indicates that the crystals belong to an orthorhombic space group, with unitcell parameters of a = 94.634, b = 156.516, c = 147.878 Å, and α = β = γ = 90°. The DCS complex crystal has slightly different unit-
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Fig. 1. Multiple sequence alignment of seven isozymes of alanine racemase. Strictly conserved residues are indicated by (⁎); highly conserved residues are indicated by (:). The two catalytic residues are boxed. Sources and protein accession numbers are as follows: E. faecalis, Q837J0; G. stearothermophilus, P10724; P. aeruginosa, Q9HTQ2; H. influenzae, P45257; E. coli, P0A6B4; H. pylori, Q1XG01; and M. tuberculosis, P0A4X2.
cell parameters with a = 94.597, b = 156.186, c = 146.930. The space group was C2221 for both crystals, determined on the basis of systematic absences. The merged data set is 97.9% complete at a resolution of 2.5 Å for the apoenzyme and 95.7% for the DCS complex.
Assuming the presence of three molecules per asymmetric unit, the calculated Matthews coefficient (VM) value is 2.26 Å3 Da− 1 [20]. The solvent content of the crystal was calculated to be 45.5%. Data refinement statistics are presented in Table 1.
A. Priyadarshi et al. / Biochimica et Biophysica Acta 1794 (2009) 1030–1040 Table 2 Conserved catalytic residues of various AlaR enzymes and their equivalent position in seven pathogenically important AlaR enzymes. AlaR source
Catalytic residues
AlaR (E. faecalis) AlaR (G. stearothermophilus) AlaR (H. influenzae) AlaR (E. coli) AlaR (P. aeruginosa) AlaR (M. tuberculosis) AlaR (H. pylori)
Lys40 Lys39 Lys36 Lys34 Lys33 Lys42 Lys37
Tyr267 Tyr265 Tyr256 Tyr255 Tyr253 Tyr271 Tyr271
The EfAlaR structure was solved by molecular replacement with CNS [16], using a monomer of the G. stearothermophilus AlaR structure (PDB ID 1EPV). The final models of the apoenzyme and the DCS complex, validated with PROCHECK[18], show that 100% of the residues lying within the allowed regions of the Ramachandran plot. For the DCS-inhibited structure, PROCHECK determined that 84.9% of all the residues were in the most favored regions of the Ramachandran plot, 14.8% of the residues were in the additionally allowed regions, and 0.3% of the residues were in the generously allowed regions. For the apoenzyme structure, PROCHECK determined that 84.3% of all the residues were in the most favored region of the Ramachandran plot, 14.3% of the residues were in the additionally allowed regions, and 1.4% of the residues were in the generously allowed regions. No residues were found in the disallowed regions of either structure. 3.3. Overall structure of the alanine racemase apoenzyme from E. faecalis The EfAlaR monomer is composed of two domains, an Nterminal and a C-terminal domain. The N-terminal domain, consisting of residues 1–229, forms a TIM barrel structure, which provides space for the active site. The N-terminal domain of EfAlaR is composed of nine α-helices (α 1–9), eight β-strands (β2–9), a PLP cofactor, and acetate. The first β-strand of the monomer is located at the extreme N-terminus of the EfAlaR structure, and does not contribute to the formation of the TIM barrel. Residues 247–371 constitute the second domain (C-terminal) of EfAlaR, and are almost completely composed of β-strands. Residues Leu230– Ala246, form a loop that connects the N-terminal with the Cterminal domain (Fig. 2). The overall structure of EfAlaR reveals that it is a homodimer in which two monomers form a head-to-tail association, such that the C-terminal domain of one monomer interacts with the N-terminal TIM barrel of the opposite monomer (Fig. 3). The EfAlaR homodimer is comprised of two active sites that catalyzed by two conserved residues Lys40 and Tyr267′ (from the other monomer) respectively. In the EfAlaR structure, a PLP cofactor binds covalently via an imine bond to the -amino group of the side chain of each Lys40, forming an internal aldimine (Schiff's base) form. The EfAlaR dimer is formed by a few polar interactions and several hydrophobic interactions, which play an important role in structure stabilization. The asymmetrical unit of the crystal consists of three molecules of EfAlaR (Fig. 3), but only the AC dimer represents the active conformation. There are approximately 17% (2900 Å2) surface contact areas (mostly hydrophobic interactions) between the A and C subunit; 72 residues of subunit A interact with 69 residues of subunit C (Fig. 4). In this EfAlaR structure, hydrophobic interactions play an important role in maintaining the dimer structure. The A and B subunits are just symmetric molecules in the crystal, as there are no effective interactions between them that would contribute to enzymatic activity. The dimer interface is composed primarily of residues located on various loops that connect the α-helices with the β-strands of the N-terminal domain
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of the first monomer, as well as loops connecting the β-strands of the C-terminal domain of the second monomer. Three active site residues participate at the dimer interface, Arg139, His169 and Tyr267′ (from opposite monomer). Each monomer binds to one PLP and acetate molecule in the active site, which is located towards the center of the TIM barrel. One PEG and one HEPES molecule bind along the surface of the dimer interface, between the A and C subunits. PEG and HEPES were included during the crystallization of EfAlaR. Interestingly, several water molecules were present at the dimer interface, making hydrogen bonds with residues and with other water molecules. 3.4. Comparison of the overall folding of alanine racemases Since 1997, AlaR structures from ten species of bacteria have been deposited in the RCSB Protein Data Bank. To elucidate the important features of this enzyme, AlaR structures from three important pathogens, AlaR from G. stearothermophilus (PDB ID 1SFT) [21], DadX from P. aeruginosa (PDB ID 1RCQ) [22], and AlaR from M. tuberculosis (PDB ID 1XFC) [23], were compared with EfAlaR. When we superimposed these AlaR structures with EfAlaR, we established that the C-terminal domain is well conserved among the four structures, but the N-terminal domain shows some variability (Fig. 5). The r.m.s. deviations between EfAlaR and the Gram-positive G. stearothermophilus AlaR (GsAlaR) and M. tuberculosis AlaR (MtAlaR) are 0.920 and 1.346, respectively, whereas, the r.m.s. deviation between EfAlaR and the Gram-negative P. aeruginosa DadX (PaDadX) is 1.924. All the eubacteria mentioned above possess alanine racemases for peptidoglycan biosynthesis. However, some bacteria, such as P. aeruginosa, possess additional catabolic alanine racemases that are distinct from other AlaR [24]. The secondary structure and
Fig. 2. Monomer structure of EfAlaR. The N-terminal domain has a TIM barrel structure composed of nine α-helices (α 1–9) and eight β-strand (β2–9). The first βstrand of the monomer is located at the extreme end of the N-terminus of the protein and does not contribute to the TIM barrel. The C-terminal domain is mostly composed of β-strands. PLP (pyridoxal 5′-phosphate) and Ace (acetate) are located near the TIM barrel.
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Fig. 3. Overall structure of EfAlaR in an asymmetrical unit. Three subunits are shown in cyan, yellow and green. Subunits A and C form the active dimer. PLP (pyridoxal 5′-phosphate), Ace (acetate), PEG (polyethylene glycol), and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) are shown at the dimer interface.
overall folding of EfAlaR is very similar to that observed for GsAlaR, MtAlaR and PaDadX, confirming a strong architectural resemblance between these prokaryotic enzymes. The regions near the active site and dimer interface superimpose very well between the EfAlaR and GsAlaR structures. Minor structural
differences exist in areas that do not participate in dimerization. In contrast, only one domain of PaDadX can be superimposed on the EfAlaR structure. Whereas the N-terminal domain is well superimposed, but, the C-terminal domain of PaDadX is rotated 15° relative to that of EfAlaR (Fig. 5).
Fig. 4. Interacting residues (buried region) between subunits A and C of EfAlaR show mostly hydrophobic interactions. The electrostatic potential mapped on-to the molecular surface of the EfAlaR dimer interface is shown separately. The electrostatic potential was computed with PYMOL.
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Fig. 5. Superposition of various alanine racemases. AlaR from Enterococcus faecalis (EfAlaR) is shown in cyan, AlaR from Geobacillus stearothermophilus (PDB ID 1EPV) in pink, DadX from Pseudomonas aeruginosa (PDB ID 1RCQ) in yellow, and AlaR from Mycobacterium tuberculosis (PDB ID 1XFC) in green. The N-terminus shows structural variability between these four AlaR structures, and the C-terminus of PaDadX is tilted 15° relative to the EfAlaR axis.
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two different monomers (Fig. 6b), i.e., residues Asp174, Glu175, Ile176, Asn231, Ser233, Lys236, Ile354 and His355 from the first monomer, and residues Tyr267′, Tyr286′, Arg292′ and Arg311′ from the second partner monomer. Among these residues, Asp174, Pro232, Tyr356, Tyr267′, Tyr286′, Arg292′ and Arg311′ are highly conserved across AlaR (Fig. 1). By investigating the charges of the residues around the entryway, we discovered that the two residues with negative charges, namely Asp174, Glu175, are positioned almost opposite the Tyr267′ residue from the partner monomer (Fig. 6b). The electrostatic surface charges around the substrate entryways of EfAlaR, and its homologs, PaDadX, GsAlaR and MtAlaR, were found to be similar. The substrate entry corridor is about ∼10.0 Å long and leads towards the reactive atom C4′of PLP. In the EfAlaR structure, the opening width of the active site corridor between Tyr356 and Tyr267′ is 6.4 Å, wider than that between the corresponding tyrosine residues in the active site of PaDadX [22] (i.e. 2.7 Å), and this increases accessibility to the substrate (van der Waals radius of 3.4 Å). Asp174, which is fully conserved across AlaR homologs (Fig. 1), forms a middle gate with Arg292′, whereas the inner gate is formed by Tyr267′ and Tyr356 near the active site pocket. Alanine racemases use several types of interactions to stabilize the substrate carboxylate group. In EfAlaR, the side chains of Arg139, and to some extent of Tyr267′, and in GsAlaR the main chain of Met 314′ [29] and Tyr354 [30], play important roles in stabilization. A conserved residue, Arg222, is unprotonated throughout the catalytic cycle because of its interaction with pyridine N. A point mutant of this residue, R222E, exhibits a decrease in kcat values of up to three fold [6], revealing that this residue is also important for stabilizing AlaR.
3.5. Active site and substrate entryway
3.6. PEG and HEPES molecules
The geometry of the active site's binding pocket (6.0 × 4.0 × 3.0 Å) of EfAlaR and of other reported AlaR structures are quite similar. In the EfAlaR structure, two residues function as the catalytic acid and base, with the Lys40 residue attacking the α-hydrogen of L-alanine to form a carbanionic intermediate, and the Tyr267′ donating a proton from the opposite side, generating the enantiomeric product, consistent with previous reports [25,26]. The conserved active site of EfAlaR, composed of these two residues along with a hydrogen-bonded network of residues, is shown in Fig. 6a. This hydrogen-bonded network plays an important role in the reaction mechanism, as noted in the description of previous AlaR structures [10,12,21,22,23]. The active site is solvent-accessible, one water molecule forms H-bonds with the oxygen atom of PLP (2.8 Å) and two additional H-bonds with the oxygen atoms from the peptide backbone or side chains of His169 and Ala208. The alanine racemization reaction is initiated via Schiff base formation between the substrate and cofactor (PLP). The substrate Rcarbon is then deprotonated and stabilized by electron delocalization via the electrophilic pyridinium nitrogen. We observed that each PLP is near the center of the TIM barrel and closer to the C-terminal domain of the partner monomer. In the EfAlaR structure, PLP concomitantly interacts with the Tyr44, Arg139, Arg222 and Tyr356 residues (Fig. 6a). The conjugated π system of the PLP ring is the functional group that reacts with the α-carbon of the substrate and plays a pivotal role in AlaR activity [27,28]. We found that the conserved Arg139 and Met314′ residues are positioned such that they are capable of forming hydrogen bonds with the carbonyl oxygen of PLP, suggesting that the NH1 of Arg139 and the main chain nitrogen of Met314′ form the recognition site for the carboxyl group of the substrate and help to stabilize AlaR. In the EfAlaR structure, the residues responsible for instantly shaping the substrate entryway are represented by residues on the
The overall structure of EfAlaR is similar to that of GsAlaR, with the exception that EfAlaR has some additional electron density on the dimer interface. After repeated refinement, one PEG molecule and one HEPES molecule were revealed in the EfAlaR structure (Fig. 7a). The PEG molecule is located on the interface of the dimer, near the His293 and His293′ residues. The PEG molecule surrounds the His293 residue and lies partly between the two His residues (His293 and His293′), whereas the HEPES molecule is located on the opposite side of His293 (Fig. 7b). Based on the primary sequence alignment (Fig. 1), these two histidine residues in EfAlaR are replaced by arginine residues in the GsAlaR. The distance between PEG and its surrounding residues, His293 (3.8 Å), Lys350 (3.9 Å), Glu352 (3.5 Å), His293′ (3.1 Å), Arg311′ (3.90 Å), and Lys359′ (3.50 Å) reveals the hydrophilic nature of the compound (Fig. 7c). PEG was found to bind to the interface site, and not the catalytic residues, but they are positioned between the two active sites and the substrate entry site with approx. distance of 4.6 Å from entryway. Thus, this molecule could affect the catalytic activity of EfAlaR. Given the potential location and properties of this PEG binding pocket, this region could be used for designing inhibitors to gain better selectivity in the future. 3.7. Structure of the EfAlaR–DCS complex We determined the crystal structure of the DCS complex to 2.5 Å resolution. The presence of DCS in the active site does not induce any significant conformational changes in the enzyme. The r.m.s. deviation between the apoenzyme structure and the DCS complex structure is 0.32 Å. Our EfAlaR structure shows that the DCS is held in the active site by a network of hydrogen bonds, which are quite similar to those found in all the structures reported for this enzyme [10,12]. A
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Fig. 6. Active site residues and the substrate entry channel of EfAlaR. (a) Hydrogen bond network of the EfAlaR active site. Residues marked with (') belong to the partner monomer. Hydrogen bonds with an interatomic distance of less than 3.0 Å are depicted by dotted lines. (b) Electrostatic surface map around the substrate entry channel, residues from the two different monomers are shown in cyan and yellow, with PEG lying between two His293 residues.
carboxylate-binding motif has been observed interacting with the hydroxy-imine that forms part of the hydroxy-isoxazole ring. The hydroxyl moiety of DCS interacts with Tyr44, Arg139, Ser207, Arg222, and Tyr356 (Fig. 8a). The residues near DCS make a hydrophilic groove that enables them to form a hydrogen bond, which helps to stabilize EfAlaR (Fig. 8b). The electron density of the conserved Tyr267, which is approximately 3.0 Å distant from DCS, was found ordered in the EfAlaR–DCS complex structure, but was disordered in the GsAlaR structure (Tyr265 in GsAlaR) reported by Fenn et al. [12]. The PLP derivatives bound to cycloserine enantiomers may be identical, as observed in GsAlaR. About eleven residues were found to interact with DCS; among them, highly conserved residues, such as Tyr44, Ser207, Arg222 and Tyr356, were
less than 3.0 Å away from DCS (Table 3). One water molecule interacts with DCS. 3.8. Structural comparisons between the EfAlaR apoenzyme and the EfAlaR–DCS complex When we superposed the apoenzyme EfAlaR structure with that of the EfAlaR–DCS complex structure, no significant conformational changes were detected (Fig. 9). The spatial positions of the amino acid side chains near PLP and DCS are almost identical (Fig. 9). However, some corresponding regions of PLP and DCS cannot be superimposed due to conformational differences between the N1 of PLP and DCS. Active site residues were found in the same positions in
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Fig. 7. Ribbon diagram of the EfAlaR dimer. (a) PEG and HEPES molecules bound to EfAlaR. (b) Residues near PEG. Residues marked with (′) belongs to the partner monomer. (c) Electronegativity map of EfAlaR. PLP, pyridoxal 5′-phosphate; Ace, acetate; PEG, polyethylene glycol; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.
the apoenzyme and DCS complex structure; however, slight conformational changes occurred in the His169 and Arg222 side chains of subunit A and in the Tyr267′ and Met314′ side chains of subunit C
(Fig. 9). In the DCS complex structure, Arg139 from subunit A moves approximately 1.5 Å towards the DCS molecule as compared to the apoenzyme EfAlaR structure (Fig. 9). The average temperature factor
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Fig. 8. Residues of EfAlaR interacting with the D-cyloserine inhibitor. (a) Diagrammatic view of σA-weighted simulated annealing omit maps of DCS. Maps are shown at 1.5σ for the DCS adduct. This figure was generated with CNS xtal and CCP4 mapcover. (b) Electrostatic surface map around DCS.
for all atoms in both structures is approximately 35 Å2; however, almost double B-factor values were observed for the loop region Gln117–Asn124 in the DCS complex (Gln117–Arg128 in apoenzyme EfAlaR). Whereas, high B-factor regions, Pro145–Gln156 and Glu274– Glu277 of the apoenzyme are absent in EfAlaR–DCS complex structure. In the apoenzyme EfAlaR structure, an extra α-helix (α12) in subunit C and an extended β-strand (β4) in subunit A were observed (Fig. 9) that were not present in the EfAlaR–DCS complex. 4. Discussion AlaR is an attractive target for antibacterial drugs because of its absence in humans and its indispensable function in bacteria.
In retrospect of drug design many substrate analogues and inhibitors were discovered for the AlaR. Two natural antibiotics (D-cycloserine and O-carbamoyl-D-serine) and several alanine analogues (β,β,β-trifluoroalanine, alanine phosphonate, 1-aminocyclopropane phosphonate, and β- chloro- and β-fluoroalanine) are commonly used to inhibit AlaR. Though a combination of Xray crystallographic and spectroscopic methods was used to decipher the mechanisms of inhibition, it was found that these inhibitors have a fatal drawback, generating secondary-effect. They act not only on AlaR but also on all PLP-dependent enzymes, several of which are expressed in humans. Therefore, a detailed structural analysis of AlaR is needed in order to develop drugs with greater specificity.
A. Priyadarshi et al. / Biochimica et Biophysica Acta 1794 (2009) 1030–1040 Table 3 D-cycloserine interactions with the peptide backbone or side chains of various residues (polar interactions for hydrogen bonding and salt bridges). Residue (side chain)
D-cycloserine
Distance (Å)
VAL38 (CG1) LYS40 (CE) TYR44 (OH) LEU86 (CD1) ARG139 (NH1) HIS169 (ND1) ASN206 (CB) SER207 (OG) ARG222 (NE) VAL225 (CG2) TYR267 (OH) TYR356 (OH)
DCS DCS DCS DCS DCS DCS DCS DCS DCS DCS DCS DCS
3.59 3.63 2.56 3.73 3.12 3.48 3.59 2.50 2.97 3.72 3.06 2.60
(C5A) (C4A) (O2P) (C2) (O3) (C2) (O1P) (O1P) (N1) (O2P) (N) (O3P)
The structure of E. faecalis alanine racemase was solved by Xray crystallography for the apoenzyme and the DCS complex. The structure shows that the peptide backbone (Cα) positions of the apoenzyme and the DCS complex are quite similar. The conserved residue Tyr267 is constrained in the EfAlaR–DCS complex structure, which was found, more flexible in the GsAlaR structure. The PLP derivatives bound to cycloserine enantiomers were noticed with the same orientation as observed in the GsAlaR structure. From EfAlaR structures, we identified a different site that may be suitable for drug targeting, i.e., the PEG binding site. PEG was bound to His293 and His293′ along the dimer interface of AlaR, where the head of the PEG molecule was oriented approximately 4.6 Å away from the substrate entry pathway. This binding may affect the structural stability and activity of AlaR. To determine the affinity of PEG for EfAlaR we performed an isothermal calorimetry (ITC) experiment. However, repeated attempts of this experiment at various protein and PEG concentrations showed only very weak or no binding. These preliminary results will need to be confirmed by further experiments (using BIACORE or another technique). The substrate entryway is a key feature of AlaR, and has been proposed
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to be a potential target for drug design, since it consists of highly conserved residues. We predict that the negatively charged residues, Asp174 and Glu175, around the substrate entryway, play an important role in substrate orientation along with the cooperation of the positively charged Arg292′ and Arg311′ from the partner monomer. 5. Conclusion The EfAlaR structure is very similar to the alanine racemase structures reported from G. stearothermophilus, M. tuberculosis, and P. aeruginosa. While the hinge angles between the monomer domains of all four structures are uniquely different, but key active site residues superimpose well. Analysis of the EfAlaR structure confirms that this enzyme remains a challenging but important target for drug design. The most significant result of this structural report involves the entryway that leads to the active site. Our analysis reveals that the inner and middle regions of the protein are strongly conserved across species, while regions near the enzyme surface are more variable. AlaR has often been proposed to be a candidate target for designing antimicrobial drugs, given its essential role in prokaryotes but absence from eukaryotes. We can design AlaR inhibitors using various approaches, such as placing an inhibitor within the active site or blocking accessibility to the active site using an inhibitor that binds to the substrate entryway, or by designing inhibitors that would prevent dimerization. Based on the study of E. faecalis, which possesses resistance to cephalosporins and penicillin, the crystal structure of EfAlaR will aid in structurebased drug design. Acknowledgments We thank Dr. H. S. Lee and his staff for assistance during data collection at beamline 4A of Pohang Light Source, Korea. A. Priyadarshi and E. E Kim were supported by KIST grants. This study was supported by the Functional Proteomics Center, 21C Frontier Program of the Korea Ministry of Science and Technology.
Fig. 9. Superposition of D-cycloserine and PLP. D-cycloserine is shown in yellow and PLP in green. The enlarged β4 motif and the additional α12 observed in the apoenzyme EfAlaR structure are shown in green, and the EfAlaR–DCS complex is shown in cyan.
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