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Alanine racemase (Alr) catalyzes the pyridoxal 5′-phosphate (PLP)-dependent racemization between l - and d -alanine in bacteria. Owing to the potential interest in targeting Alr for antibacterial drug development, several studies have determined the structures of Alr from different species, proposing models for the reaction mechanism. Insights into its reaction dynamics may be conducive to a better understanding of the Alr reaction mechanism. In this study, we determined the structures of the apo and reaction states of Bacillus subtilis Alr (BsAlr) at room temperature using a fixed-target based X-ray free-electron laser. The 2.3 Å resolution structures revealed the alanine substrate or intermediate in various positions at the active site. Conformational change between the N- and C-terminal domains of BsAlr expanded the entryway for substrate binding. In the reaction state of BsAlr, two main alanine binding states were observed: one alanine molecule is positioned away from PLP, whereas the other alanine molecule is covalently bonded to PLP. These structures might represent the dynamic states of the substrate for entrance into, reaction with, or exit from the active site. Our approach provides a simple and rapid method for elucidating the intermediate structure of Alr, which can be expanded to other enzymes.

Whole-cell screening for Mycobacterium tuberculosis ( Mtb ) inhibitors is complicated by the pathogen’s slow growth and biocontainment requirements. Here we present a synthetic biology framework for assaying Mtb drug targets in engineered E. coli . We construct Target Essential Surrogate E. coli (TESEC) in which an essential metabolic enzyme is deleted and replaced with an Mtb -derived functional analog, linking bacterial growth to the activity of the target enzyme. High throughput screening of a TESEC model for Mtb alanine racemase (Alr) revealed benazepril as a targeted inhibitor, a result validated in whole-cell Mtb . In vitro biochemical assays indicated a noncompetitive mechanism unlike that of clinical Alr inhibitors. We establish the scalability of TESEC for drug discovery by characterizing TESEC strains for four additional targets. Whole-cell screening for Mycobacterium tuberculosis inhibitors is complicated by the pathogen’s slow growth and biocontainment requirements. Here the authors develop engineered E. coli as a synthetic biology tool to express and screen metabolic targets from Mycobacterium tuberculosis .

Pyridoxal phosphate (PLP) is an enzyme cofactor required for the chemical transformation of biological amines in many central cellular processes. PLP-dependent enzymes (PLP-DEs) are ubiquitous and evolutionarily diverse, making their classification based on sequence homology challenging. Here we present a chemical proteomic method for reporting on PLP-DEs using functionalized cofactor probes. We synthesized pyridoxal analogues modified at the 2′-position, which are taken up by cells and metabolized in situ. These pyridoxal analogues are phosphorylated to functional cofactor surrogates by cellular pyridoxal kinases and bind to PLP-DEs via an aldimine bond which can be rendered irreversible by NaBH4 reduction. Conjugation to a reporter tag enables the subsequent identification of PLP-DEs using quantitative, label-free mass spectrometry. Using these probes we accessed a significant portion of the Staphylococcus aureus PLP-DE proteome (73%) and annotate uncharacterized proteins as novel PLP-DEs. We also show that this approach can be used to study structural tolerance within PLP-DE active sites and to screen for off-targets of the PLP-DE inhibitor d-cycloserine.

In an effort to discover new drugs to treat tuberculosis (TB) we chose alanine racemase as the target of our drug discovery efforts. In Mycobacterium tuberculosis, the causative agent of TB, alanine racemase plays an essential role in cell wall synthesis as it racemizes L-alanine into D-alanine, a key building block in the biosynthesis of peptidoglycan. Good antimicrobial effects have been achieved by inhibition of this enzyme with suicide substrates, but the clinical utility of this class of inhibitors is limited due to their lack of target specificity and toxicity. Therefore, inhibitors that are not substrate analogs and that act through different mechanisms of enzyme inhibition are necessary for therapeutic development for this drug target. To obtain non-substrate alanine racemase inhibitors, we developed a high-throughput screening platform and screened 53,000 small molecule compounds for enzyme-specific inhibitors. We examined the 'hits' for structural novelty, antimicrobial activity against M. tuberculosis, general cellular cytotoxicity, and mechanism of enzyme inhibition. We identified seventeen novel non-substrate alanine racemase inhibitors that are structurally different than any currently known enzyme inhibitors. Seven of these are active against M. tuberculosis and minimally cytotoxic against mammalian cells. This study highlights the feasibility of obtaining novel alanine racemase inhibitor lead compounds by high-throughput screening for development of new anti-TB agents.

is a pathogenic and zoonotic bacteria that impacted high mortality to many fish species as well as capable of causing serious disease to humans. Alanine racemase (Alr, EC 5.1.1.1) is a pyridoxal-5’-phosphate (PLP)-containing homodimeric enzyme that catalyzes the racemization of L-alanine and D-alanine. In this study, we purified alanine racemase from that was isolated from an infected Chinese sturgeon ( ), as well as determined its biochemical characteristics and inhibitors. The gene has an open reading frame (ORF) of 1107 bp, encoding a protein of 369 amino acids, which has a molecular mass of 40 kDa. The enzyme has optimal activity at a temperature of 35°C and a pH of 9.5. It belongs to the PLP-dependent enzymes family and is highly specific to L-alanine. Alr (SiAlr) could be inhibited by some metal ions, hydroxylamine and dithiothreitol (DTT). The kinetic parameters and of the enzyme were 33.11 mM, 2426 units/mg for L-alanine, and 14.36 mM, 963.6 units/mg for D-alanine. Finally, the 50% inhibitory concentrations (IC ) values and antibiotic activity of two alanine racemase inhibitors (homogentisic acid and hydroquinone), were determined and found to be effective against both Gram-positive and Gram-negative bacteria employed in this study.

Pyridoxal 5'-phosphate (PLP) dependent alanine racemase catalyzes racemization of L-Ala to D-Ala, a key component of the peptidoglycan network in bacterial cell wall. It has been extensively studied as an important antimicrobial drug target due to its restriction in eukaryotes. However, many marketed alanine racemase inhibitors also act on eukaryotic PLP-dependent enzymes and cause side effects. A thermostable alanine racemase (AlrTt) from Thermoanaerobacter tengcongensis MB4 contains an evolutionarily non-conserved residue Gln360 in inner layer of the substrate entryway, which is supposed to be a key determinant in substrate specificity. Here we determined the crystal structure of AlrTt in complex with L-Ala at 2.7 Å resolution, and investigated the role of Gln360 by saturation mutagenesis and kinetic analysis. Compared to typical bacterial alanine racemase, presence of Gln360 and conformational changes of active site residues disrupted the hydrogen bonding interactions necessary for proper PLP immobilization, and decreased both the substrate affinity and turnover number of AlrTt. However, it could be complemented by introduction of hydrophobic amino acids at Gln360, through steric blocking and interactions with a hydrophobic patch near active site pocket. These observations explained the low racemase activity of AlrTt, revealed the essential role of Gln360 in substrate selection, and its preference for hydrophobic amino acids especially Tyr in bacterial alanine racemization. Our work will contribute new insights into the alanine racemization mechanism for antimicrobial drug development.

Threonine aldolases catalyze the pyridoxal phosphate (PLP) dependent cleavage of threonine into glycine and acetaldehyde and play a major role in the degradation of this amino acid. In nature, L- as well as D-specific enzymes have been identified, but the exact physiological function of D-threonine aldolases (DTAs) is still largely unknown. Both types of enantio-complementary enzymes have a considerable potential in biocatalysis for the stereospecific synthesis of various β-hydroxy amino acids, which are valuable building blocks for the production of pharmaceuticals. While several structures of L-threonine aldolases (LTAs) have already been determined, no structure of a DTA is available to date. Here, we report on the determination of the crystal structure of the DTA from Alcaligenes xylosoxidans (AxDTA) at 1.5 Å resolution. Our results underline the close relationship of DTAs and alanine racemases and allow the identification of a metal binding site close to the PLP-cofactor in the active site of the enzyme which is consistent with the previous observation that divalent cations are essential for DTA activity. Modeling of AxDTA substrate complexes provides a rationale for this metal dependence and indicates that binding of the β-hydroxy group of the substrate to the metal ion very likely activates this group and facilitates its deprotonation by His193. An equivalent involvement of a metal ion has been implicated in the mechanism of a serine dehydratase, which harbors a metal ion binding site in the vicinity of the PLP cofactor at the same position as in DTA. The structure of AxDTA is completely different to available structures of LTAs. The enantio-complementarity of DTAs and LTAs can be explained by an approximate mirror symmetry of crucial active site residues relative to the PLP-cofactor.

The L-alanine mediated germination of food isolated Bacillus cereus DSA 1 spores, which lacked an intact exosporium, increased in the presence of D-cycloserine (DCS), which is an alanine racemase (Alr) inhibitor, reflecting the activity of the Alr enzyme, capable of converting L-alanine to the germination inhibitor D-alanine. Proteomic analysis of the alkaline extracts of the spore proteins, which include exosporium and coat proteins, confirmed that Alr was present in the B. cereus DSA 1 spores and matched to that encoded by B. cereus ATCC 14579, whose spore germination was strongly affected by the block of conversion of L- to D-alanine. Unlike ATCC 14579 spores, L-alanine germination of B. cereus DSA 1 spores was not affected by the preincubation with DCS, suggesting a lack of restriction in the reactant accessibility.

Burkholderia pseudomallei and Burkholderia mallei are category B select agents and must be studied under BSL3 containment in the United States. They are typically resistant to multiple antibiotics, and the antibiotics used to treat B. pseudomallei or B. mallei infections may not be used as selective agents with the corresponding Burkholderia species. Here, we investigated alanine racemase deficient mutants of B. pseudomallei and B. mallei for development of non-antibiotic-based genetic selection methods and for attenuation of virulence. The genome of B. pseudomallei K96243 has two annotated alanine racemase genes (bpsl2179 and bpss0711), and B. mallei ATCC 23344 has one (bma1575). Each of these genes encodes a functional enzyme that can complement the alanine racemase deficiency of Escherichia coli strain ALA1. Herein, we show that B. pseudomallei with in-frame deletions in both bpsl2179 and bpss0711, or B. mallei with an in-frame deletion in bma1575, requires exogenous D-alanine for growth. Introduction of bpsl2179 on a multicopy plasmid into alanine racemase deficient variants of either Burkholderia species eliminated the requirement for D-alanine. During log phase growth without D-alanine, the viable counts of alanine racemase deficient mutants of B. pseudomallei and B. mallei decreased within 2 hours by about 1000-fold and 10-fold, respectively, and no viable bacteria were present at 24 hours. We constructed several genetic tools with bpsl2179 as a selectable genetic marker, and we used them without any antibiotic selection to construct an in-frame ΔflgK mutant in the alanine racemase deficient variant of B. pseudomallei K96243. In murine peritoneal macrophages, wild type B. mallei ATCC 23344 was killed much more rapidly than wild type B. pseudomallei K96243. In addition, the alanine racemase deficient mutant of B. pseudomallei K96243 exhibited attenuation versus its isogenic parental strain with respect to growth and survival in murine peritoneal macrophages.

A thermostable alanine racemase from Thermoanaerobacter tengcongensis MB4 was successfully expressed in Escherichia coli and characterized. The full-length gene MBalr2 (1164 bp) encodes 388 amino acid residues including 6 out of 8 highly conserved amino acid residues at the entryway to the active site of alanine racemase. Recombinant MBAlr2 and three mutants (S171A, H359Y and double mutation S171A/H359Y) of MBAlr2 were purified by His 6 -tag affinity column and gel filtration chromatography. The purified protein MBAlr2 was a dimeric PLP-dependent enzyme with broad substrate specificity. The optimal racemization temperature and pH were 70–75 °C and 11.0, respectively. The kinetic parameters K m and V max of MBAlr2 at 70 °C, determined by HPLC, were 20.16 mM and 1414 μmol min −1 for l -alanine, and 9.95 mM and 702.6 μmol min −1 for d -alanine, respectively. Enzymatic assays showed that the activity of both mutants (S171A and H359Y) was lost, but the activity of mutant S171A/H359Y was recovered to 69.8 % of wild type, which suggested that residues Ser171 and His359 might be the important residues for catalytic mechanisms of MBAlr2.