RESUMEN
Synergistic interactions between chemical inhibitors, whilst informative, can be difficult to interpret, as chemical inhibitors can often have multiple targets, many of which can be unknown. Here, using multiplexed transcriptional repression, we have validated that the simultaneous repression of glutamate racemase and alanine racemase has a synergistic interaction in Mycobacterium tuberculosis. This confirms prior observations from chemical interaction studies and highlights the potential of targeting multiple enzymes involved in mycobacterial cell wall synthesis.
Asunto(s)
Alanina Racemasa , Isomerasas de Aminoácido , Mycobacterium tuberculosis , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/efectos de los fármacos , Isomerasas de Aminoácido/genética , Isomerasas de Aminoácido/metabolismo , Alanina Racemasa/genética , Alanina Racemasa/metabolismo , Regulación Bacteriana de la Expresión Génica , Transcripción Genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Inhibidores Enzimáticos/farmacología , Pared Celular/metabolismo , Pared Celular/genéticaRESUMEN
Although bacterial peptidoglycan (PG) is highly conserved, some natural variations in PG biosynthesis and structure have evolved. Understanding the mechanisms and limits of such variation will inform our understanding of antibiotic resistance, innate immunity, and the evolution of bacteria. We have explored the constraints on PG evolution by blocking essential steps in PG biosynthesis in Vibrio fischeri and then selecting mutants with restored prototrophy. Here, we attempted to select prototrophic suppressors of a D-glutamate auxotrophic murI racD mutant. No suppressors were isolated on unsupplemented lysogeny broth salts (LBS), despite plating >1011 cells, nor were any suppressors generated through mutagenesis with ethyl methanesulfonate. A single suppressor was isolated on LBS supplemented with iso-D-gln, although the iso-D-gln subsequently appeared irrelevant. This suppressor has a genomic amplification formed by the creation of a novel junction that fuses proB to a gene encoding a putative broad-spectrum racemase of V. fischeri, bsrF. An engineered bsrF allele lacking the putative secretion signal (ΔSS-bsrF) also suppressed D-glu auxotrophy, resulting in PG that was indistinguishable from the wild type. The ΔSS-bsrF allele similarly suppressed the D-alanine auxotrophy of an alr mutant and restored prototrophy to a murI alr double mutant auxotrophic for both D-ala and D-glu. The ΔSS-bsrF allele increased resistance to D-cycloserine but had no effect on sensitivity to PG-targeting antibiotics penicillin, ampicillin, or vancomycin. Our work helps define constraints on PG evolution and reveals a periplasmic broad-spectrum racemase in V. fischeri that can be co-opted for PG biosynthesis, with concomitant D-cycloserine resistance. IMPORTANCE: D-Amino acids are used and produced by organisms across all domains of life, but often, their origins and roles are not well understood. In bacteria, D-ala and D-glu are structural components of the canonical peptidoglycan cell wall and are generated by dedicated racemases Alr and MurI, respectively. The more recent discovery of additional bacterial racemases is broadening our view and deepening our understanding of D-amino acid metabolism. Here, while exploring alternative PG biosynthetic pathways in Vibrio fischeri, we unexpectedly shed light on an unusual racemase, BsrF. Our results illustrate a novel mechanism for the evolution of antibiotic resistance and provide a new avenue for exploring the roles of non-canonical racemases and D-amino acids in bacteria.
Asunto(s)
Alanina Racemasa , Ácido Glutámico , Ácido Glutámico/metabolismo , Aliivibrio fischeri/genética , Aliivibrio fischeri/metabolismo , Racemasas y Epimerasas/metabolismo , Cicloserina , Peptidoglicano/metabolismo , Aminoácidos/metabolismo , Alanina Racemasa/metabolismoRESUMEN
Brucella, a zoonotic facultative intracellular pathogenic bacterium, poses a significant threat both to human health and to the development of the livestock industry. Alanine racemase (Alr), the enzyme responsible for alanine racemization, plays a pivotal role in regulating virulence in this bacterium. Moreover, Brucella mutants with alr gene deletions (Δalr) exhibit potential as vaccine candidates. However, the mechanisms that underlie the detrimental effects of alr knockouts on Brucella pathogenicity remain elusive. Here, initially, we conducted a bioinformatics analysis of Alr, which demonstrated a high degree of conservation of the protein within Brucella spp. Subsequent metabolomics studies unveiled alterations in amino acid pathways following deletion of the alr gene. Furthermore, alr deletion in Brucella suis S2 induced decreased resistance to stress, antibiotics, and other factors. Transmission electron microscopy of simulated macrophage intracellular infection revealed damage to the cell wall in the Δalr strain, whereas propidium iodide staining and alkaline phosphatase and lactate dehydrogenase assays demonstrated alterations in cell membrane permeability. Changes in cell wall properties were revealed by measurements of cell surface hydrophobicity and zeta potential. Finally, the diminished adhesion capacity of the Δalr strain was shown by immunofluorescence and bacterial enumeration assays. In summary, our findings indicate that the alr gene that regulates amino acid metabolism in Brucella influences the properties of the cell wall, which modulates bacterial adherence capability. This study is the first demonstration that Alr impacts virulence by modulating bacterial metabolism, thereby providing novel insights into the pathogenic mechanisms of Brucella spp.
Asunto(s)
Alanina Racemasa , Brucella , Brucelosis , Humanos , Alanina Racemasa/genética , Alanina Racemasa/química , Alanina Racemasa/metabolismo , Brucella/metabolismo , Antibacterianos , Pared Celular/metabolismo , AminoácidosRESUMEN
Brucella suis, the causative agent of brucellosis, poses a significant public health and animal husbandry threat. However, the role of the alanine racemase (alr) gene, which encodes alanine racemase in Brucella, remains unclear. Here, we analyzed an alr deletion mutant and a complemented strain of Brucella suis S2. The knockout strain displayed an unaltered, smooth phenotype in acriflavine agglutination tests but lacked the core polysaccharide portion of lipopolysaccharide (LPS). Genes involved in the LPS synthesis were significantly upregulated in the deletion mutant. The alr deletion strain exhibited reduced intracellular viability in the macrophages, increased macrophage-mediated killing, and upregulation of the apoptosis markers. Bcl2, an anti-apoptotic protein, was downregulated, while the pro-apoptotic proteins, Bax, Caspase-9, and Caspase-3, were upregulated in the macrophages infected with the deletion strain. The infected macrophages showed increased mitochondrial membrane permeability, Cytochrome C release, and reactive oxygen species, activating the mitochondrial apoptosis pathway. These findings revealed that alanine racemase was dispensable in B. suis S2 but influenced the strain's rough features and triggered the mitochondrial apoptosis pathway during macrophage invasion. The deletion of the alr gene reduced the intracellular survival and virulence. This study enhances our understanding of the molecular mechanism underlying Brucella's survival and virulence and, specifically, how alr gene affects host immune evasion by regulating bacterial LPS biosynthesis.
Asunto(s)
Alanina Racemasa , Brucella suis , Brucelosis , Animales , Brucella suis/genética , Lipopolisacáridos , Virulencia/genética , Brucelosis/microbiologíaRESUMEN
Eradication of Helicobacter pylori, the class 1 carcinogen, faces several obstacles, which demand alternative options to conventional drug development methods. Alanine racemase (Alr) was proposed as H. pylori drug target, inhibited by propanoic acid (PA), in a previous in silico study. We investigated the possible treatment of H. pylori infection through Alr inhibition. A new model of H. pylori Alr was built, validated, and the binding of PA to the active site was modelled via molecular docking with a good docking score. PA minimum inhibitory concentration (MIC) against H. pylori ATCC 43504 and six H. pylori clinical isolates ranged from 312.5 to 416.7 ± 180 µg/ml and remained unchanged after 14 serial passages in increasing PA concentrations. The minimum bactericidal concentration of PA was 625 µg/ml. Selective Alr inhibition was confirmed by a significant PA MIC increase with increasing d-alanine concentrations. Similar PA MIC in other tested pathogens was recorded (312.5-625 µg/ml). PA lacked cytotoxicity in tested cell lines and efficiently eradicated H. pylori in a rat infection model. In conclusion, Alr is a promising broad-spectrum drug target, inhibited by PA without resistance development by repeated exposure for 14 serial passages.
Asunto(s)
Alanina Racemasa , Infecciones por Helicobacter , Helicobacter pylori , Ratas , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Alanina Racemasa/química , Simulación del Acoplamiento Molecular , Propionatos/uso terapéutico , Infecciones por Helicobacter/tratamiento farmacológico , Pruebas de Sensibilidad MicrobianaRESUMEN
Given the ever-present threat of antibacterial resistance, there is an urgent need to identify new antibacterial drugs and targets. One such target is alanine racemase (Alr), an enzyme required for bacterial cell-wall biosynthesis. Alr is an attractive drug target because it is essential for bacterial survival but is absent in humans. Existing drugs targeting Alr lack specificity and have severe side effects. We here investigate alternative mechanisms of Alr inhibition. Alr functions exclusively as an obligate homodimer, so we probed seven conserved interactions on the dimer interface, distant from the enzymatic active site, to identify possible allosteric influences on activity. Using the Alr from Mycobacterium tuberculosis (MT) as a model, we found that the Lys261/Asp135 salt bridge is critical for catalytic activity. The Lys261Ala mutation completely inactivated the enzyme, and the Asp135Ala mutation reduced catalytic activity eight-fold. Further investigation suggested a potential drug-binding site near the Lys261/Asp135 salt bridge that may be useful for allosteric drug discovery.
Asunto(s)
Alanina Racemasa , Mycobacterium tuberculosis , Humanos , Antibacterianos/farmacología , Alanina Racemasa/genética , Alanina Racemasa/química , Alanina Racemasa/metabolismo , Dominio Catalítico , Mycobacterium tuberculosis/genética , Farmacorresistencia BacterianaRESUMEN
Alanine racemases (ALRs) are essential for d-alanine (d-Ala) production in bacteria, and many ALRs have a conserved carbamylated lysine residue in the active site. Although short-chain carboxylates inhibit ALRs harbouring this lysine residue as substrate analogues, in an ALR variant with an alanine residue at this position, carboxylates behave as activators; however, this activation mechanism remains unclear. Here, we performed kinetic and structural analyses of U1ALR, an ALR from Latilactobacillus sakei UONUMA harbouring a glycine residue (Gly134) in the site of the carbamylated lysine residue. U1ALR was activated by various carboxylates and also by a G134K mutation, both of which caused a significant decrease in Km , indicating an increase in substrate affinity. The U1ALR crystal structure revealed the presence of an acetate molecule bound in a position and at an orientation resembling the conformation of the carbamylated lysine side chain observed in the structures of other ALRs. These results suggest a regulatory mechanism for U1ALR activity involving two carboxylate-binding sites: one with high affinity near Gly134, where an acetate molecule is observed in the crystal structure and carboxylate binding results in enzyme activation; the other is the substrate-binding site, where carboxylate binding inhibits enzyme activity. Furthermore, we observed no carboxylate/G134K-mediated activation in the presence of d-Ala at high concentrations, implying that d-Ala also exhibits low-affinity binding in the first carboxylate-binding site and prevents carboxylate/G134K-induced activation. Such regulation of enzyme activity by carboxylates and d-Ala may be ubiquitous in many ALRs from lactic acid bacteria sharing the same sequence characteristics.
Asunto(s)
Alanina Racemasa , Alanina Racemasa/genética , Alanina Racemasa/química , Alanina Racemasa/metabolismo , Alanina/genética , Alanina/metabolismo , Lisina , Sitios de Unión , Dominio Catalítico , Ácidos Carboxílicos , CinéticaRESUMEN
The Enterobacterial Rcs stress response system reacts to envelope stresses through a complex two-component phosphorelay system to regulate a variety of environmental response genes, such as capsular polysaccharide and flagella biosynthesis genes. However, beyond Escherichia coli, the stresses that activate Rcs are not well-understood. In this study, we used a Rcs system-dependent luminescent transcriptional reporter to screen a library of over 240 antimicrobial compounds for those that activated the Rcs system in Serratia marcescens, a Yersiniaceae family bacterium. Using an isogenic rcsB mutant to establish specificity, both new and expected activators were identified, including the short-chain fatty acid propionic acid, which is found at millimolar levels in the human gut. Propionic acid did not reduce the bacterial intracellular pH, as was hypothesized for its antibacterial mechanism. Instead, data suggest that the Rcs-activation by propionic acid is due, in part, to an inactivation of alanine racemase. This enzyme is responsible for the biosynthesis of d-alanine, which is an amino-acid that is required for the generation of bacterial cell walls. Consistent with what was observed in S. marcescens, in E. coli, alanine racemase mutants demonstrated elevated expression of the Rcs-reporter in a d-alanine-dependent and RcsB-dependent manner. These results suggest that host gut short-chain fatty acids can influence bacterial behavior via the activation of the Rcs stress response system. IMPORTANCE The Rcs bacterial stress response system responds to envelope stresses by globally altering gene expression to profoundly impact host-pathogen interactions, virulence, and antibiotic tolerance. In this study, a luminescent Rcs-reporter plasmid was used to screen a library of compounds for activators of Rcs. Among the strongest inducers was the short-chain fatty acid propionic acid, which is found at high concentrations in the human gut. This study suggests that gut short-chain fatty acids can affect both bacterial virulence and antibiotic tolerance via the induction of the Rcs system.
Asunto(s)
Alanina Racemasa , Proteínas de Escherichia coli , Alanina/metabolismo , Alanina Racemasa/genética , Alanina Racemasa/metabolismo , Antibacterianos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Propionatos/farmacología , Propionatos/metabolismoRESUMEN
Alanine racemase is a pyridoxal-5'-phosphate dependent bacterial enzyme that provides the essential peptidoglycan precursor D-alanine, utilized for cell wall synthesis. This enzyme is ubiquitous throughout bacteria, including Mycobacterium tuberculosis, making it an attractive target for antibacterial drug discovery. We investigated the binding mode of twenty five reported Mycobacterium tuberculosis alanine racemase inhibitors. The results obtained from molecular docking studies emphasized the importance of inhibitor interaction with Lys42, Tyr46, Arg140, His172 and Tyr175 residues at the catalytic binding pocket of alanine racemase enzyme. The predicted binding free energies showed that van der Waals and nonpolar solvation interactions are the driving force for binding of inhibitors. Molecular dynamics simulation studies of four such inhibitor-alanine racemase systems were further explored to study the inhibition mechanism. The quantum chemical parameters calculated at the B3LYP/6-31G**++ level of theory indicated that the inhibitors must have low values of the lowest unoccupied molecular orbital energy and high values of electrostatic potential for stronger interactions. We expect that this study can provide significant theoretical guidance for design of potent Mycobacterium tuberculosis alanine racemase inhibitors in future.
Asunto(s)
Alanina Racemasa , Mycobacterium tuberculosis , Alanina/química , Alanina Racemasa/química , Alanina Racemasa/metabolismo , Antibacterianos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Mycobacterium tuberculosis/metabolismoRESUMEN
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.
Asunto(s)
Alanina Racemasa , Mycobacterium tuberculosis , Alanina Racemasa/química , Alanina Racemasa/metabolismo , Antituberculosos/química , Antituberculosos/farmacología , Descubrimiento de Drogas , Escherichia coli/genética , Escherichia coli/metabolismo , Ensayos Analíticos de Alto Rendimiento , Mycobacterium tuberculosis/metabolismoRESUMEN
The hyperthermophilic bacterium Thermotoga maritima has an atypical peptidoglycan that contains d-lysine alongside the usual d-alanine and d-glutamate. We previously identified a lysine racemase involved in d-lysine biosynthesis, and this enzyme also possesses alanine racemase activity. However, T. maritima has neither alanine racemase nor glutamate racemase enzymes; hence, the precise biosynthetic pathways of d-alanine and d-glutamate remain unclear in T. maritima. In the present study, we identified and characterized a novel d-amino acid aminotransferase (TM0831) in T. maritima. TM0831 exhibited aminotransferase activity towards 23 d-amino acids, but did not display activity towards l-amino acids. It displayed high specific activities towards d-homoserine and d-glutamine as amino donors. The most preferred acceptor was 2-oxoglutarate, followed by glyoxylate. Additionally, TM0831 displayed racemase activity towards four amino acids including aspartate and glutamate. Catalytic efficiency (kcat /Km ) for aminotransferase activity was higher than for racemase activity, and pH profiles were distinct between these two activities. To evaluate the functions of TM0831, we constructed a TTHA1643 (encoding glutamate racemase)-deficient Thermus thermophilus strain (∆TTHA1643) and integrated the TM0831 gene into the genome of ∆TTHA1643. The growth of this TM0831-integrated strain was promoted compared with ∆TTHA1643 and was restored to almost the same level as that of the wild-type strain. These results suggest that TM0831 is involved in d-glutamate production. TM0831 is a novel d-amino acid aminotransferase with racemase activity that is involved in the production of d-amino acids in T. maritima.
Asunto(s)
Alanina Racemasa , Aminoácidos , Alanina/genética , Alanina/metabolismo , Alanina Racemasa/metabolismo , Aminoácidos/metabolismo , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Vías Biosintéticas , Ácido Glutámico/metabolismo , Glutamina/metabolismo , Glioxilatos , Homoserina/metabolismo , Ácidos Cetoglutáricos , Lisina/genética , Lisina/metabolismo , Peptidoglicano/metabolismo , Thermotoga maritima/genética , Transaminasas/genética , Transaminasas/metabolismoRESUMEN
Alanine racemase (EC 5.1.1.1) depends on pyridoxal 5'-phosphate and catalyses the interconversion between L- and D-Ala. The enzyme is responsible for the biosynthesis of D-Ala, which is an essential component of the peptidoglycan layer of bacterial cell walls. Phylogenetic analysis of alanine racemases demonstrated that the cyanobacterial enzyme diverged before the separation of gram-positive and gram-negative enzymes. This result is interesting considering that the peptidoglycans observed in cyanobacteria seem to combine the properties of those in both gram-negative and gram-positive bacteria. We cloned the putative alanine racemase gene (slr0823) of Synechocystis sp. PCC6803 in Escherichia coli cells, expressed and purified the enzyme protein and studied its enzymological properties. The enzymatic properties of the Synechocystis enzyme were similar to those of other gram-positive and gram-negative bacterial enzymes. Alignment of the amino acid sequences of alanine racemase enzymes revealed that the conserved tyrosine residue in the active centre of most of the gram-positive and gram-negative bacterial enzymes has been replaced with tryptophan in most of the cyanobacterial enzymes. We carried out the site-directed mutagenesis involving the corresponding residue of Synechocystis enzyme (W385) and revealed that the residue is involved in the substrate recognition by the enzyme.
Asunto(s)
Alanina Racemasa , Synechocystis , Alanina/genética , Alanina Racemasa/química , Alanina Racemasa/genética , Alanina Racemasa/metabolismo , Secuencia de Aminoácidos , Mutagénesis Sitio-Dirigida , Filogenia , Synechocystis/genética , Synechocystis/metabolismoRESUMEN
An enzymatic assay system of d-Ala, which is reported to affect the taste, was constructed using alanine racemase and l-alanine dehydrogenase. d-Ala is converted to l-Ala by alanine racemase and then deaminated by l-alanine dehydrogenase with the reduction of NAD+ to NADH, which is determined with water-soluble tetrazolium. Using the assay system, the d-Ala contents of 7 crustaceans were determined.
Asunto(s)
Alanina RacemasaRESUMEN
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.
Asunto(s)
Bacillus subtilis/crecimiento & desarrollo , Biopelículas/crecimiento & desarrollo , Simbiosis , Alanina Racemasa/genética , Alanina Racemasa/metabolismo , Bacillus subtilis/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Medios de Cultivo , Regulación Bacteriana de la Expresión Génica , Técnicas de Silenciamiento del GenRESUMEN
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.
Asunto(s)
Alanina Racemasa/metabolismo , Antibióticos Antituberculosos/química , Cicloserina/química , Proteínas Recombinantes/metabolismo , Alanina/química , Alanina/metabolismo , Alanina Racemasa/genética , Secuencia de Aminoácidos , Antibióticos Antituberculosos/metabolismo , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cicloserina/metabolismo , Escherichia coli , Isoxazoles/química , Ligasas/metabolismo , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Oximas/química , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/genéticaRESUMEN
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.
Asunto(s)
Alanina Racemasa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Oligopéptidos/química , Oligopéptidos/farmacología , Tetrazoles/química , Alanina Racemasa/metabolismo , Inhibidores Enzimáticos/química , Pruebas de Sensibilidad Microbiana , Oligopéptidos/síntesis química , Técnicas de Síntesis en Fase SólidaRESUMEN
Streptococcus iniae 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 S. iniae that was isolated from an infected Chinese sturgeon (Acipenser sinensis), as well as determined its biochemical characteristics and inhibitors. The alr 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. S. iniae Alr (SiAlr) could be inhibited by some metal ions, hydroxylamine and dithiothreitol (DTT). The kinetic parameters K m and V max 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 (IC50) 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.Streptococcus iniae 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 S. iniae that was isolated from an infected Chinese sturgeon (Acipenser sinensis), as well as determined its biochemical characteristics and inhibitors. The alr 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. S. iniae Alr (SiAlr) could be inhibited by some metal ions, hydroxylamine and dithiothreitol (DTT). The kinetic parameters K m and V max 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 (IC50) 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.
Asunto(s)
Alanina Racemasa/química , Alanina Racemasa/aislamiento & purificación , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Inhibidores Enzimáticos/química , Infecciones Estreptocócicas/microbiología , Streptococcus iniae/enzimología , Alanina Racemasa/antagonistas & inhibidores , Alanina Racemasa/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Estabilidad de Enzimas , Humanos , Cinética , Filogenia , Alineación de Secuencia , Streptococcus iniae/química , Especificidad por SustratoRESUMEN
Pyridoxal 5'-phosphate (PLP) is a versatile cofactor involved in a large variety of enzymatic processes. Most of PLP-catalysed reactions, such as those of alanine racemases (AlaRs), present a common resting state in which the PLP is covalently bound to an active-site lysine to form an internal aldimine. The crystal structure of BsAlaR grown in the presence of Tris lacks this covalent linkage and the PLP cofactor appears deformylated. However, loss of activity in a Tris buffer only occurred after the solution was frozen prior to carrying out the enzymatic assay. This evidence strongly suggests that Tris can access the active site at subzero temperatures and behave as an alternate racemase substrate leading to mechanism-based enzyme inactivation, a hypothesis that is supported by additional X-ray structures and theoretical results from QM/MM calculations. Taken together, our findings highlight a possibly underappreciated role for a common buffer component widely used in biochemical and biophysical experiments.
Asunto(s)
Alanina Racemasa/metabolismo , Bacillus subtilis/enzimología , Frío , Iminas/metabolismo , Alanina Racemasa/química , Alanina Racemasa/aislamiento & purificación , Iminas/química , Modelos Moleculares , Estructura Molecular , Teoría CuánticaRESUMEN
BACKGROUND: Aeromonas hydrophila is an opportunistic pathogen of poikilothermic and homoeothermic animals, including humans. In the present study, we described the role of Alanine racemase (alr-2) in the virulence of A. hydrophila using an alr-2 knockout mutant (A.H.Δalr). RESULTS: In mouse and common carp models, the survival of animals challenged with A.H.Δalr was significantly increased compared with the wild-type (WT), and the mutant was also impaired in its ability to replicate in the organs and blood of infected mice and fish. The A.H.Δalr significantly increased phagocytosis by macrophages of the mice and fish. These attenuation effects of alr-2 could be complemented by the addition of D-alanine to the A.H.Δalr strain. The histopathology results indicated that the extent of tissue injury in the WT-infected animals was more severe than in the A.H.Δalr-infected groups. The expression of 9 virulence genes was significantly down-regulated, and 3 outer membrane genes were significantly up-regulated in A.H.Δalr. CONCLUSIONS: Our data suggest that alr-2 is essential for the virulence of A. hydrophila. Our findings suggested alanine racemase could be applied in the development of new antibiotics against A. hydrophila.
Asunto(s)
Aeromonas hydrophila/genética , Aeromonas hydrophila/patogenicidad , Alanina Racemasa/genética , Técnicas de Inactivación de Genes , Factores de Virulencia/genética , Aeromonas hydrophila/enzimología , Animales , Proteínas Bacterianas/genética , Carpas/microbiología , Femenino , Infecciones por Bacterias Gramnegativas/microbiología , Ratones Endogámicos BALB C , Mutación , Virulencia/genéticaRESUMEN
Clostridium perfringens is a gram-positive, anaerobic, pathogenic bacterium that can cause a wide range of diseases in humans, poultry and agriculturally important livestock. A pyridoxal-5-phosphate-dependent alanine racemase with a function in the racemization of d- and l-alanine is an attractive drug target for C. perfringens and other pathogens due to its absence in animals and humans. In this study alanine racemase from C. perfringens (CPAlr) was successfully expressed and purified in Escherichia coli and biochemically characterized. The purified CPAlr protein was a dimeric PLP-dependent enzyme with high substrate specificity. The optimal racemization temperature and pH were 40°C and 8.0, respectively. The kinetic parameters Km and kcat of CPAlr, determined by HPLC at 40°C were 19.1 mM and 17.2 s-1 for l-alanine, and 10.5 mM and 8.7 s-1 for d-alanine, respectively. Gel filtration chromatographic analysis showed that the molecular weight of mutant Y359A was close to monomeric form, suggesting that the inner layer residue Tyr359 might play an essential role in dimer-formation. Furthermore, the mutation at residues Asp171 and Tyr359 resulted in a dramatic increase in Km value and/or decreased in kcat value, indicating that the middle and inner layer residues Asp171 and Tyr359 of CPAlr might have the key role in substrate binding, catalytic activity or oligomerization state through the hydrogen-bonding interaction with the pentagonal ring waters and/or PLP cofactor.