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The broad-spectrum antibiotic d -cycloserine (DCS) is a key component of regimens used to treat multi- and extensively drug-resistant tuberculosis. DCS, a structural analog of d -alanine, binds to and inactivates two essential enzymes involved in peptidoglycan biosynthesis, alanine racemase (Alr) and d -Ala: d -Ala ligase. Inactivation of Alr is thought to proceed via a mechanism-based irreversible route, forming an adduct with the pyridoxal 5′-phosphate cofactor, leading to bacterial death. Inconsistent with this hypothesis, Mycobacterium tuberculosis Alr activity can be detected after exposure to clinically relevant DCS concentrations. To address this paradox, we investigated the chemical mechanism of Alr inhibition by DCS. Inhibition of M. tuberculosis Alr and other Alrs is reversible, mechanistically revealed by a previously unidentified DCS-adduct hydrolysis. Dissociation and subsequent rearrangement to a stable substituted oxime explains Alr reactivation in the cellular milieu. This knowledge provides a novel route for discovery of improved Alr inhibitors against M. tuberculosis and other bacteria. d -Cycloserine inactivates alanine racemase by forming an adduct with the pyridoxal 5′-phosphate cofactor, but structural and spectroscopic analyses reveal that reactivation occurs on adduct hydrolysis and product rearrangement to a stable oxime.

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 .

Life in a three-dimensional biofilm is typical for many bacteria, yet little is known about how strains interact in this context. Here, we created essential gene CRISPR interference knockdown libraries in biofilm-forming Bacillus subtilis and measured competitive fitness during colony co-culture with wild type. Partial knockdown of some translation-related genes reduced growth rates and led to out-competition. Media composition led some knockdowns to compete differentially as biofilm versus non-biofilm colonies. Cells depleted for the alanine racemase AlrA died in monoculture but survived in a biofilm colony co-culture via nutrient sharing. Rescue was enhanced in biofilm colony co-culture with a matrix-deficient parent due to a mutualism involving nutrient and matrix sharing. We identified several examples of mutualism involving matrix sharing that occurred in three-dimensional biofilm colonies but not when cultured in two dimensions. Thus, growth in a three-dimensional colony can promote genetic diversity through sharing of secreted factors and may drive evolution of mutualistic behavior.

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.

Amino acids play essential roles in various biological processes. In humans, most amino acids are present in the l -form; however, small amounts of d -amino acids also exist and have significant physiological roles, highlighting the importance of dietary intake from foods or drinks. In this study, we investigated the amino acid composition of the geoduck clam Panopea japonica , emphasizing its remarkably high d -alanine ( d -Ala) content in the siphon tissue. The d -Ala content (6.99–14.2 mmol/100 g-wet) amounted to 91–94% of the total Ala, far exceeding that of other bivalves such as Tresus keenae (74%). Enzyme assays revealed alanine racemase and d -amino acid oxidase activities, suggesting active d -Ala biosynthesis and metabolism. The high concentrations of d -Ala enhance its value as a delicacy owing to its unique sweetness. This study provides new insights into the biosynthesis and metabolic characteristics of d -Ala in bivalves, highlighting its physiological and food ingredient significance.

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.

d-tagatose is a popular functional monosaccharide produced from lactose by β-galactosidase and arabinose isomerase. In this study, two d-alanine-deficient heterologous gene expression systems were constructed, B. subtilis 168 D1 and B. subtilis 168 D2, using overlapping extension PCR and the CRE/loxP system. The lacZ gene for β-galactosidase was integrated into a specific locus of the chassis B. subtilis 168 D2. A mutually complementary plasmid pMA5 with the alanine racemase gene alrA attached to it was constructed and used to assemble recombinant plasmids overexpressing β-galactosidase and arabinose isomerase. Afterward, an integrated recombinant was constructed by the plasmid expressing the arabinose isomerase gene araA of E. coli transform-competent B. subtilis 168 D2 cells. The co-expressing plasmids were introduced into alanine racemase knockout B. subtilis 168 D1. Whole-cell bioconversion was performed using the integrated recombinant with a maximum yield of 96.8 g/L d-tagatose from 500 g/L lactose, and the highest molar conversions were 57.2%. B. subtilis 168 D1/pMA5-alrA-araA-lacZ is capable of single-cell one-step production of d-tagatose. This study provides a new approach to the production of functional sugars.

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.

In clinical culture media inoculated with patient samples, selective inhibition of commensal bacteria is essential for accurate diagnosis and effective treatment, as they can mask the presence of pathogenic bacteria. The alanine analogue, 1-aminoethyltetrazole was investigated as a potential alanine racemase inhibitor. For effective uptake and enhanced and selective antibacterial activity, a library of C-terminal 1-aminoethyltetrazole containing di- and oligopeptides were synthesized by solid phase peptide coupling techniques. The investigation of the antimicrobial activity of the synthesised compounds identified several clinically applicable selective inhibitors. These enabled differentiation between the closely related bacteria, Salmonella and Escherichia coli, which can be difficult to discriminate between in a clinical setting. In addition, differentiation between enterococci and other Gram-positive cocci was also seen.