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N ω -hydroxy-l-arginine amidinohydrolase (EC:3.5.3.25), an enzyme in the d-cycloserine (d-CS) biosynthetic pathway of Streptomyces lavendulae, catalyzes the hydrolysis of an arginase inhibitor, N ω -hydroxy-l-arginine, to produce l-ornithine and hydroxyurea, despite being homologous to arginase. Like arginase, the enzyme (DcsB) possesses two manganese ions (Mn A and Mn B ) essential for the enzymatic reaction at the bottom of the cavity formed within the molecule. However, one of the Mn A ligands in DcsB is Cys86, whereas the corresponding residues in arginase are histidine. In this study, we determined the crystal structure of Mn-free DcsB to elucidate the installation mechanism of the manganese ions. The flipping of the His111 residue after the formation of the coordination bond to the second manganese ion may facilitate the installation of Mn B and the closing of the cavity entrance to retain Mn A and Mn B at the active site. Copper ions, which are known to be a positive regulator of many secondary metabolites in Streptomyces species, were found to irreversibly inactivate the catalytic activity of DcsB. Mass spectrometric and crystallographic analyses of the Cu(II)-treated DcsB indicated that Cys86 is oxidized to sulfinic acid. The d-CS biosynthesis in the producing microorganism may be negatively regulated by the concentration of intracellular copper ions, which mediates the oxidative stress., (© 2024 Federation of European Biochemical Societies.)

Tuberculosis (TB) is the second leading cause of death by a single infectious disease behind COVID-19. Despite a century of effort, the current TB vaccine does not effectively prevent pulmonary TB, promote herd immunity, or prevent transmission. Therefore, alternative approaches are needed. We seek to develop a cell therapy that produces an effective antibiotic in response to TB infection. D-cycloserine (D-CS) is a second-line antibiotic for TB that inhibits bacterial cell wall synthesis. We have determined D-CS to be the optimal candidate for anti-TB cell therapy due to its effectiveness against TB, relatively short biosynthetic pathway, and its low-resistance incidence. The first committed step towards D-CS synthesis is catalyzed by the L-serine-O-acetyltransferase (DcsE) which converts L-serine and acetyl-CoA to O-acetyl-L-serine (L-OAS). To test if the D-CS pathway could be an effective prophylaxis for TB, we endeavored to express functional DcsE in A549 cells as a human pulmonary model. We observed DcsE-FLAG-GFP expression using fluorescence microscopy. DcsE purified from A549 cells catalyzed the synthesis of L-OAS as observed by HPLC-MS. Therefore, human cells synthesize functional DcsE capable of converting L-serine and acetyl-CoA to L-OAS demonstrating the first step towards D-CS production in human cells., (© 2023. The Author(s).)

The O-ureidoserine racemase (DcsC) is an enzyme found from the biosynthetic gene cluster of antitubercular agent d-cycloserine. Although DcsC is homologous to diaminopimelate epimerase (DapF) that catalyzes the interconversion between ll- and dl-diaminopimelic acid, it specifically catalyzes the interconversion between O-ureido-l-serine and its enantiomer. Here we determined the crystal structure of DcsC at a resolution of 2.12 Å, implicating that the catalytic mechanism of DcsC shares similarity with that of DapF. Comparing the structure of the active center of DcsC to that of DapF, Thr72, Thr198, and Tyr219 of DcsC are likely to be involved in the substrate specificity., (© 2021 Wiley Periodicals LLC.)

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 (DCS) and amantadine (AMA) act as partial NMDA receptor (R) agonist and antagonist, respectively. In the present study, we compared the effects of DCS and AMA on dopamine D 2/3 R binding in the brain of adult rats in relation to motor behavior. D 2/3 R binding was determined with small animal SPECT in baseline and after challenge with DCS (20 mg/kg) or AMA (40 mg/kg) with [ 123 I]IBZM as radioligand. Immediately post-challenge, motor/exploratory behavior was assessed for 30 min in an open field. The regional binding potentials (ratios of the specifically bound compartments to the cerebellar reference region) were computed in baseline and post-challenge. DCS increased D 2/3 R binding in nucleus accumbens, substantia nigra/ventral tegmental area, thalamus, frontal, motor and parietal cortex as well as anterodorsal and posterior hippocampus, whereas AMA decreased D 2/3 R binding in nucleus accumbens, caudateputamen and thalamus. After DCS, ambulation and head-shoulder motility were decreased, while sitting was increased compared to vehicle and AMA. Moreover, DCS increased rearing relative to AMA. The regional elevations of D 2/3 R binding after DCS reflect a reduction of available dopamine throughout the mesolimbocortical system. In contrast, the reductions of D 2/3 R binding after AMA indicate increased dopamine in nucleus accumbens, caudateputamen and thalamus. Findings imply that, after DCS, nigrostriatal and mesolimbic dopamine levels are directly related to motor/exploratory activity, whereas an inverse relationship may be inferred for AMA.

Background and Objectives: The dosing of cycloserine and terizidone is the same, as both drugs are considered equivalent or used interchangeably. Nevertheless, it is not certain from the literature that these drugs are interchangeable. Therefore, the amount of cycloserine resulting from the metabolism of terizidone and the relationship with hepatic function were determined., Methods: This prospective clinical study involved 39 patients with drug-resistant tuberculosis admitted for an intensive phase of treatment. Cycloserine pharmacokinetic parameters for individual patients, like area under the curve (AUC), clearance (CLm/F), peak concentration (C max ) and trough concentration (C min ), were calculated from a previously validated joint population pharmacokinetic model of terizidone and cycloserine. Correlation and regression analyses were performed for pharmacokinetic parameters and unconjugated bilirubin (UB), conjugated bilirubin (CB), albumin, the ratio of aspartate transaminase to alanine aminotransferase (AST/ALT), or binding affinity of UB to albumin (K af ), using R statistical software version 3.5.3., Results: Thirty-eight patients took a daily dose of 750 mg terizidone, while one took 500 mg. The amount of cycloserine [median (range)] that emanated from terizidone metabolism was 51.6 (0.64-374) mg. C max (R 2  = 22%, p = 0.003) and C min (R 2  = 10.6%, p = 0.044) were significantly associated with increased CB concentration. C max was significantly associated with increased K af (R 2  = 10.1%, p = 0.048), while high CLm/F was significantly associated with decreased AST/ALT (R 2  = 21%, p = 0.003)., Conclusions: Cycloserine is not interchangeable with terizidone, as amounts are lower than expected. Cycloserine may be a predisposing factor to the development of hyperbilirubinaemia, as CLm/F is affected by hepatic function.

D-cycloserine is an antibiotic which targets sequential bacterial cell wall peptidoglycan biosynthesis enzymes: alanine racemase and D-alanine:D-alanine ligase. By a combination of structural, chemical and mechanistic studies here we show that the inhibition of D-alanine:D-alanine ligase by the antibiotic D-cycloserine proceeds via a distinct phosphorylated form of the drug. This mechanistic insight reveals a bimodal mechanism of action for a single antibiotic on different enzyme targets and has significance for the design of future inhibitor molecules based on this chemical structure.

Previously, we successfully cloned a d-cycloserine (d-CS) biosynthetic gene cluster consisting of 10 open reading frames (designated dcsA to dcsJ) from d-CS-producing Streptomyces lavendulae ATCC 11924. In this study, we put four d-CS biosynthetic genes (dcsC, dcsD, dcsE, and dcsG) in tandem under the control of the T7 promoter in an Escherichia coli host. SDS-PAGE analysis demonstrated that the 4 gene products were simultaneously expressed in host cells. When l-serine and hydroxyurea (HU), the precursors of d-CS, were incubated together with the E. coli resting cell suspension, the cells produced significant amounts of d-CS (350 ± 20 μM). To increase the productivity of d-CS, the dcsJ gene, which might be responsible for the d-CS excretion, was connected downstream of the four genes. The E. coli resting cells harboring the five genes produced d-CS at 660 ± 31 μM. The dcsD gene product, DcsD, forms O-ureido-l-serine from O-acetyl-l-serine (OAS) and HU, which are intermediates in d-CS biosynthesis. DcsD also catalyzes the formation of l-cysteine from OAS and H2S. To repress the side catalytic activity of DcsD, the E. coli chromosomal cysJ and cysK genes, encoding the sulfite reductase α subunit and OAS sulfhydrylase, respectively, were disrupted. When resting cells of the double-knockout mutant harboring the four d-CS biosynthetic genes, together with dcsJ, were incubated with l-serine and HU, the d-CS production was 980 ± 57 μM, which is comparable to that of d-CS-producing S. lavendulae ATCC 11924 (930 ± 36 μM)., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: We have recently been successful in cloning a gene cluster necessary for the biosynthesis of D-cycloserine (D-CS) from D-CS-producing Streptomyces lavendulae ATCC11924. Although dcsD, one of the ORFs located on the gene cluster, encodes a protein homologous to O-acetylserine sulfhydrylase that synthesizes L-cysteine using O-acetyl-L-serine together with sulfide, it functions to form O-ureido-L-serine as a D-CS biosynthetic intermediate, using O-acetyl-L-serine together with hydroxyurea (HU). In the present study, using crystallographic and mutational studies, three amino acid residues in DcsD that are important for the substrate preference toward HU were determined. We showed that two of the three residues are important for the binding of HU into the substrate-binding pocket. The other residue contributes to the formation of a loose hydrogen-bond network during the catalytic reaction. Information regarding the amino acid residues will be very useful in the design of a new catalyst for synthesizing the β-substituted-L-alanine derivatives., Database: The atomic coordinates and structure factors of wild-type DcsD and l-OUS-bound K43A mutant of DcsD have been deposited in the Protein Data Bank under accession codes 3X43 and 3X44, respectively., (© 2015 FEBS.)

Introduction: Tuberculosis remains a formidable threat to global public health. Multidrug-resistant tuberculosis presents increasing burden on the control strategy. D-Cycloserine (DCS) is an effective second-line drug against Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis. Though less potent than isoniazid (INH) and streptomycin, DCS is crucial for antibiotic-resistant tuberculosis. One advantage of DCS is that less drug-resistant M. tuberculosis is reported in comparison with first-line antituberculosis drugs such as INH and rifampin., Areas Covered: In this review, we summarise our current knowledge of DCS, and review the drug target and low-level resistance of DCS in M. tuberculosis. We summarise the metabolism of D-alanine (D-Ala) and peptidoglycan biosynthesis in bacteria. We first compared the amino acid similarity of Mycobacterium alanine racemase and D-Ala:D-alanine ligase and quite unexpectedly found that the two enzymes are highly conserved among Mycobacterium., Expert Opinion: We summarise the drug targets of DCS and possible mechanisms underlying its low-level resistance for the first time. One significant finding is that ubiquinone and menaquinone metabolism-related genes are novel genes underlying DCS resistance in Escherichia coli and with homologues in M. tuberculosis. Further understanding of DCS targets and basis for its low-level resistance might inspire us to improve the use of DCS or find better drug targets.

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.

Vibrio parahaemolyticus is a halophilic Gram-negative bacterium that causes human gastroenteritis. When the viable but nonculturable (VBNC) state of this bacterium was induced by incubation at 4°C in Morita minimal salt solution containing 0.5% NaCl, the rod-shaped cells became coccoid, and various aberrantly shaped intermediates were formed in the initial stage. This study examined the factors that influence the formation of these aberrantly shaped cells. The proportion of aberrantly shaped cells was not affected in a medium containing D-cycloserine (50 μg/ml) but was lower in a medium containing cephalosporin C (10 μg/ml) than in the control medium without antibiotics. The proportion of aberrantly shaped cells was higher in a culture medium that contained 0.5% NaCl than in culture media containing 1.0 or 1.5% NaCl. The expression of 15 of 17 selected genes associated with cell wall synthesis was enhanced, and the expression of VP2468 (dacB), which encodes D-alanyl-D-alanine carboxypeptidase, was enhanced the most. The proportion of aberrantly shaped cells was significantly lower in the dacB mutant strain than in the parent strain, but the proportion was restored in the presence of the complementary dacB gene. This study suggests that disturbance of the dynamics of cell wall synthesis by enhanced expression of the VP2468 gene is associated with the formation of aberrantly shaped cells in the initial stage of induction of VBNC V. parahaemolyticus cells under specific conditions.

Cycloserine-cefoxitin fructose agar (CCFA), CCFA with horse blood and taurocholate (CCFA-HT), and cycloserine-cefoxitin mannitol broth with taurocholate and lysozyme (CCMB-TAL) were compared for recovery of Clostridium difficile from 120 stool specimens. Compared to CCFA, CCFA-HT enhanced C. difficile growth and improved recovery by 4%. In a separate study, 9% (8/91) of stool samples previously C. difficile negative on plate medium were C. difficile positive when cultured in CCMB-TAL.

We have recently cloned a DNA fragment containing a gene cluster that is responsible for the biosynthesis of an antituberculosis antibiotic, D-cycloserine. The gene cluster is composed of 10 open reading frames, designated dcsA to dcsJ. Judging from the sequence similarity between each putative gene product and known proteins, DcsC, which displays high homology to diaminopimelate epimerase, may catalyze the racemization of O-ureidoserine. DcsD is similar to O-acetylserine sulfhydrylase, which generates L-cysteine using O-acetyl-L-serine with sulfide, and therefore, DcsD may be a synthase to generate O-ureido-L-serine using O-acetyl-L-serine and hydroxyurea. DcsG, which exhibits similarity to a family of enzymes with an ATP-grasp fold, may be an ATP-dependent synthetase converting O-ureido-D-serine into D-cycloserine. In the present study, to characterize the enzymatic functions of DcsC, DcsD, and DcsG, each protein was overexpressed in Escherichia coli and purified to near homogeneity. The biochemical function of each of the reactions catalyzed by these three proteins was verified by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and, in some cases, mass spectrometry. The results from this study demonstrate that by using a mixture of the three purified enzymes and the two commercially available substrates O-acetyl-L-serine and hydroxyurea, synthesis of D-cycloserine was successfully attained. These in vitro studies yield the conclusion that DcsD and DcsG are necessary for the syntheses of O-ureido-L-serine and D-cycloserine, respectively. DcsD was also able to catalyze the synthesis of L-cysteine when sulfide was added instead of hydroxyurea. Furthermore, the present study shows that DcsG can also form other cyclic d-amino acid analogs, such as D-homocysteine thiolactone.

d-Cycloserine (DCS) is a broad-spectrum antibiotic that inhibits d-alanine ligase and alanine racemase activity. When Escherichia coli K-12 or CFT073 is grown in minimal glucose or glycerol medium, CycA transports DCS into the cell. E. coli K-12 cycA and CFT073 cycA mutant strains display increased DCS resistance when grown in minimal medium. However, the cycA mutants exhibit no change in DCS sensitivity compared to their parental strains when grown in LB (CFT073 and K-12) or human urine (CFT073 only). These data suggest that cycA does not participate in DCS sensitivity when strains are grown in a non-minimal medium. The small RNA GvcB acts as a negative regulator of E. coli K-12 cycA expression when grown in LB. Three E. coli K-12 gcvB mutant strains failed to demonstrate a change in DCS sensitivity when grown in LB. This further suggests a limited role for cycA in DCS sensitivity. To aid in the identification of E. coli genes involved in DCS sensitivity when grown on complex media, the Keio K-12 mutant collection was screened for DCS-resistant strains. dadA, pnp, ubiE, ubiF, ubiG, ubiH, and ubiX mutant strains showed elevated DCS resistance. The phenotypes associated with these mutants were used to further define three previously characterized E. coli DCS-resistant strains (χ316, χ444, and χ453) isolated by Curtiss and colleagues (R. Curtiss, III, L. J. Charamella, C. M. Berg, and P. E. Harris, J. Bacteriol. 90:1238-1250, 1965). A dadA mutation was identified in both χ444 and χ453. In addition, results are presented that indicate for the first time that DCS can antagonize d-amino acid dehydrogenase (DadA) activity.

Salmonella typhimurium DCyD (StDCyD) is a fold type II pyridoxal 5' phosphate (PLP)-dependent enzyme that catalyzes the degradation of D-Cys to H(2)S and pyruvate. It also efficiently degrades β-chloro-D-alanine (βCDA). D-Ser is a poor substrate while the enzyme is inactive with respect to L-Ser and 1-amino-1-carboxy cyclopropane (ACC). Here, we report the X-ray crystal structures of StDCyD and of crystals obtained in the presence of D-Cys, βCDA, ACC, D-Ser, L-Ser, D-cycloserine (DCS) and L-cycloserine (LCS) at resolutions ranging from 1.7 to 2.6 Å. The polypeptide fold of StDCyD consisting of a small domain (residues 48-161) and a large domain (residues 1-47 and 162-328) resembles other fold type II PLP dependent enzymes. The structures obtained in the presence of D-Cys and βCDA show the product, pyruvate, bound at a site 4.0-6.0 Å away from the active site. ACC forms an external aldimine complex while D- and L-Ser bind non-covalently suggesting that the reaction with these ligands is arrested at Cα proton abstraction and transimination steps, respectively. In the active site of StDCyD cocrystallized with DCS or LCS, electron density for a pyridoxamine phosphate (PMP) was observed. Crystals soaked in cocktail containing these ligands show density for PLP-cycloserine. Spectroscopic observations also suggest formation of PMP by the hydrolysis of cycloserines. Mutational studies suggest that Ser78 and Gln77 are key determinants of enzyme specificity and the phenolate of Tyr287 is responsible for Cα proton abstraction from D-Cys. Based on these studies, a probable mechanism for the degradation of D-Cys by StDCyD is proposed.

Clostridium perfringens has been associated with necrotizing enterocolitis (NEC), which is a serious disease of neonates. Our study describes the novel use of selective tryptose sulfite cycloserine with egg yolk agar (TSC-EYA) during a nursery outbreak. This medium provides a rapid, sensitive, and accurate presumptive identification of C. perfringens.

It has been long thought that hyperactivation of N-methyl-D-aspartate (NMDA) receptors underlies neurological decline after traumatic brain injury. However, all clinical trials with NMDA receptor antagonists failed. Since NMDA receptors are down-regulated from 4h to 2weeks after brain injury, activation at 24h, rather than inhibition, of these receptors, was previously shown to be beneficial in mice. Here, we tested the therapeutic window, dose regimen and mechanism of action of the NMDA receptor partial agonist D-cycloserine (DCS) in traumatic brain injury. Male mice were subjected to trauma using a weight-drop model, and administered 10mg/kg (i.p.) DCS or vehicle once (8, 16, 24, or 72h) twice (24 and 48h) or three times (24, 48 and 72h). Functional recovery was assessed for up to 60days, using a Neurological Severity Score that measures neurobehavioral parameters. In all groups in which treatment was begun at 24 or 72h neurobehavioral function was significantly better than in the vehicle-treated groups. Additional doses, on days 2 and 3 did not further improve recovery. Mice treated at 8h or 16h post injury did not differ from the vehicle-treated controls. Co-administration of the NMDA receptor antagonist MK-801 completely blocked the protective effect of DCS given at 24h. Infarct volume measured by 2,3,5-triphenyltetrazolium chloride staining at 48h or by cresyl violet at 28days was not affected by DCS treatment. Since DCS is used clinically for other indications, the present study offers a novel approach for treating human traumatic brain injury with a therapeutic window of at least 24h., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

The gamma-butyrolactone signaling system is distributed widely among streptomycetes as an important regulatory mechanism of antibiotic production and/or morphological differentiation. IM-2 [(2R,3R,1'R)-2-(1'-hydroxybutyl)-3-hydroxymethyl-gamma-butanolide] is a gamma-butyrolactone that switches off the production of D: -cycloserine but switches on the production of several nucleoside antibiotics as well as blue pigment in Streptomyces lavendulae FRI-5. farX is a member of the afsA-family genes, which are proposed to encode enzymes involved in gamma-butyrolactone biosynthesis. Disruption of farX caused overproduction of D: -cycloserine, and abolished production of nucleoside antibiotic and blue pigment with the loss of IM-2 production. The finding that all phenotypic changes observed in the farX disruptant were restored by the addition of exogenous IM-2 suggested that FarX plays a biosynthetic role in IM-2 production. Transcriptional comparison between the wild-type strain and the farX disruptant revealed that, in addition to already known genes farR1 and farR2, several other genes (farR4, farD, and farE) are under the transcriptional regulation of IM-2. Furthermore, the fact that farX transcription is under the control of IM-2 suggested that S. lavendulae FRI-5 has a fine-tuning system to control gamma-butyrolactone production.

Activation of the glycine modulatory site of the N-methyl-D-aspartate glutamate receptor (NMDAR) may reduce cognitive impairments associated with normal ageing. In order to test this hypothesis, we assessed the effects of the partial agonist D-cycloserine (DCS) on cellular activities involved in memory formation. This was performed in CA1 cellular networks of adult and aged Sprague-Dawley rat hippocampal slices using extracellular field excitatory postsynaptic potential recordings. Synaptic potentials specifically mediated by NMDAR were significantly reduced in aged animals. DCS increased the magnitude of these responses in both adult and old rats but this effect was significantly higher in the latter, thus reversing the age-related decrease in NMDAR synaptic potentials. NMDAR-mediated theta burst long-term potentiation (TBS-LTP) as well as long-term depression (LTD) of synaptic transmission, prominent models for the cellular basis of learning and memory, were also weakened in aged animals. Age-related alterations of both forms of synaptic plasticity were rescued by DCS. In addition, the DCS-induced decrease in basal fast glutamatergic neurotransmission involving the activation of inhibitory glycinergic receptors, previously reported in young rats (Rouaud & Billard, 2003), was severely attenuated in aged animals. In summary, our results indicate that the facilitation of NMDAR activation through its glycine-binding site rescues the age-related deficit of cellular mechanisms of learning and memory. Such physiological evidences suggest that this modulation site of NMDAR represents an important target to alleviate cognitive deficits associated with normal ageing.

d-Serine is an amino acid present in mammalian urine that is inhibitory to Escherichia coli strains lacking a functional dsdA gene. Counterintuitively, a dsdA strain of E. coli clinical isolate CFT073 hypercolonizes the bladder and kidneys of mice relative to wild type during a coinfection in the murine model of urinary tract infection. We are interested in the mechanisms for uptake of d-serine in CFT073. d-Serine enters E. coli K-12 via CycA, the d-alanine transporter and d-cycloserine sensitivity locus. CFT073 cycA can grow on minimal medium with d-serine as a sole carbon source. The dsdX gene of the dsdCXA locus is a likely candidate for an additional d-serine transporter based on its predicted amino acid sequence similarity to gluconate transporters. In minimal medium, CFT073 dsdX can grow on d-serine as a sole carbon source; however, CFT073 dsdX cycA cannot. Additionally, CFT073 dsdXA cycA is not sensitive to inhibitory concentrations of d-serine during growth on glycerol and d-serine minimal medium. d-[(14)C]serine uptake experiments with CFT073 dsdX cycA harboring dsdX or cycA recombinant plasmids confirm that d-serine is able to enter E. coli cells via CycA or DsdX. In whole-cell d-[(14)C]serine uptake experiments, DsdX has an apparent K(m) of 58.75 microM and a V(max) of 75.96 nmol/min/mg, and CycA has an apparent K(m) of 82.40 microM and a V(max) of 58.90 nmol/min/mg. Only d-threonine marginally inhibits DsdX-mediated d-serine transport, whereas d-alanine, glycine, and d-cycloserine inhibit CycA-mediated d-serine transport. DsdX or CycA is sufficient to transport physiological quantities of d-serine, but DsdX is a d-serine-specific permease.

This paper describes the use of the alr gene, encoding alanine racemase, as a promoter-screening tool for the identification of conditional promoters in Lactobacillus plantarum. Random fragments of the L. plantarum WCFS1 genome were cloned upstream of the promoterless alr gene of Lactococcus lactis in a low-copy-number plasmid vector. The resulting plasmid library was introduced into an L. plantarum Deltaalr strain (MD007), and 40,000 clones were selected. The genome coverage of the library was estimated to be 98%, based on nucleotide insert sequence and restriction analyses of the inserts of randomly selected clones. The library was screened for clones that were capable of complementing the D-alanine auxotroph phenotype of MD007 in media containing up to 10, 100, or 300 micro g of the competitive Alr inhibitor D-cycloserine per ml. Western blot analysis with polyclonal antibodies raised against lactococcal Alr revealed that the Alr production level required for growth increased in the presence of increasing concentrations of D-cycloserine, adding a quantitative factor to the primarily qualitative nature of the alr complementation screen. Screening of the alr complementation library for clones that could grow only in the presence of 0.8 M NaCl resulted in the identification of eight clones that upon Western blot analysis showed significantly higher Alr production under high-salt conditions than under low-salt conditions. These results established the effectiveness of the alanine racemase complementation screening method for the identification of promoters on their conditional or constitutive activity.

Alanine racemase (EC 5.1.1.1) catalyzes the interconversion of alanine enantiomers, and thus represents the first committed step involved in bacterial cell wall biosynthesis. Cycloserine acts as a suicide inhibitor of alanine racemase and as such, serves as an antimicrobial agent. The chemical means by which cycloserine inhibits alanine racemase is unknown. Through spectroscopic assays, we show here evidence of a pyridoxal derivative (arising from either isomer of cycloserine) saturated at the C4' carbon position. We additionally report the L- and D-cycloserine inactivated crystal structures of Bacillus stearothermophilus alanine racemase, which corroborates the spectroscopy via evidence of a 3-hydroxyisoxazole pyridoxamine derivative. Upon the basis of the kinetic and structural properties of both the L- and D-isomers of the inhibitor, we propose a mechanism of alanine racemase inactivation by cycloserine. This pathway involves an initial transamination step followed by tautomerization to form a stable aromatic adduct, a scheme similar to that seen in cycloserine inactivation of aminotransferases.

Bacterial alanine racemases are classified into two types of subunit structure (monomer and homodimer). To clarify the catalytic unit of monomeric alanine racemases, we examined the apparent molecular mass of the monomeric alanine racemases from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei by gel filtration in the presence of the substrate and inhibitor. The enzymes were eluted on gel filtration as a monomer of about 39,000 Da at low protein concentration and in the absence of L-alanine and D-cycloserine. An increase in the apparent molecular mass was induced by increasing the protein concentration or by adding the ligands in the elution buffer. The increase ratio depended on the ligand concentration, and the maximum apparent molecular masses of all enzymes were 60,000 and 76,000 Da in the presence of 100 mM L-alanine and 5 mM D-cycloserine, respectively. D-cycloserine may induce an inactive dimer and L-alanine may induce an intermediate between the monomer and dimer because of dynamic equilibrium. The apoenzyme also showed similar behavior in the presence of the ligands, but the increase ratios were lower than those of the holoenzymes. The Bacillus psychrosaccharolyticus alanine racemase, having a dimeric structure, showed a constant molecular mass irrespective of the absence or presence of the ligands. These results suggest that the monomeric Shigella Alr enzymes have a dimeric structure in the catalytic reaction. Substances that inhibit the subunit interaction of monomeric alanine racemases may be useful as a new type of antibacterial.

Stress stimuli can mediate apoptosis by generation of the lipid second messenger, ceramide. Herein we investigate the molecular mechanism of ceramide signaling in endothelial apoptosis induced by fenretinide (N-(4-hydroxyphenyl)retinamide (4-HPR)). 4-HPR, a synthetic derivative of retinoic acid that induces ceramide in tumor cell lines, has been shown to have antiangiogenic effects, but the molecular mechanism of these is largely unknown. We report that 4-HPR was cytotoxic to endothelial cells (50% cytotoxicity at 2.4 microm, 90% at 5.36 microm) and induced a caspase-dependent endothelial apoptosis. 4-HPR (5 microm) increased ceramide levels in endothelial cells 5.3-fold, and the increase in ceramide was required to achieve the apoptotic effect of 4-HPR. The 4-HPR-induced increase in ceramide was suppressed by inhibitors of ceramide synthesis, fumonisin B(1), myriocin, and l-cycloserine, and 4-HPR transiently activated serine palmitoyltransferase, demonstrating that 4-HPR induced de novo ceramide synthesis. Sphingomyelin levels were not altered by 4-HPR, and desipramine had no effect on ceramide level, suggesting that sphingomyelinase did not contribute to the 4-HPR-induced ceramide increase. Finally, the pancaspase inhibitor, t-butyloxycarbonyl-aspartyl[O-methyl]-fluoromethyl ketone, suppressed 4-HPR-mediated apoptosis but not ceramide accumulation, suggesting that ceramide is upstream of caspases. Our results provide the first evidence that increased ceramide biosynthesis is required for 4-HPR-induced endothelial apoptosis and present a molecular mechanism for its antiangiogenic effects.

Enrichment with D-cycloserine was used to identify Escherichia coli auxotrophic mutants that exhibited limited growth in human urine. Bacterial synthesis of guanine, arginine, and glutamine was found to be required for optimal growth in urine. Mutants that required leucine, methionine, serine, phenylalanine, or proline also exhibited reduced growth in urine. Several other nutritional mutants, including nicotinamide auxotrophs, which are found frequently among cystitis isolates, exhibited normal growth in urine.

A new differential and selective, bismuth-iron-sulfite-cycloserine (BISC) medium, for isolation and enumeration of Clostridium perfringens from food and feces, was developed. The medium was compared with the widely-used tryptose-sulfite-cycloserine (TSC) medium and blood agar (BA) in recovering actively growing cells, cold- (refrigerated and frozen) stressed, and heat-stressed C. perfringens cells, and heat-activated spores from human feces. Both selective media were satisfactory in recovering actively growing cells and heat-activated spores of C. perfringens. Both were inferior to non-inhibitory blood agar in recovering heat or cold-stressed cells. The advantages of the new BISC medium over the TSC medium were: elimination of the need to prepare pour- or overlay-agar plates, which simplified inoculation of specimens on the medium and simplified the subcultures of colonies for confirmatory identification. All colonies of C. perfringens developed on BISC were black or dark gray. This was contrary to TSC medium, which gave, on average, 39.6% of white colonies when inoculated with the pure cultures of C. perfringens.

Two uracil auxotrophs of the phytopathogen Burkolderia cepacia ATCC 25416, which is known to be involved in food spoilage, were isolated by a combination of ethylmethane sulphonate and D-cycloserine counterselection. One mutant exhibited depressed orotate phosphoribosyltransferase activity while the other mutant lacked orotidine 5'-monophosphate decarboxylase activity. Pyrimidine limitation of either auxotroph elevated aspartate transcarbamoylase and dihydroorotase activities by at least 1:5-fold indicating that these pathway enzymes may be repressible by a uracil-related compound in B. cepacia. Overall, regulation of de novo pyrimidine synthesis in the uracil auxotrophs of B. cepacia ATCC 25416 was observed.

1. The ability of D-cycloserine to act as a substrate for H+/amino acid symport has been tested in epithelial layers of Caco-2 human intestinal cells. 2. In Na(+)-free media with the apical bathing media held at pH 6.0, D-cycloserine (20 mM) is an effective inhibitor of net transepithelial transport (Jnet) of L-alanine (100 microM) and its accumulation (across the apical membrane) in a similar manner to amino acid substrates (L-alanine, beta-alanine, L-proline and glycine). In contrast L-valine was ineffective as an inhibitor for H+/amino acid symport. Both inhibition of L-alanine Jnet and its accumulation by D-cycloserine were dose-dependent, maximal inhibition being achieved by 5-10 mM. 3. Both D-cycloserine and known substrates for H+/amino acid symport stimulated an inward short circuit current (Isc) when voltage-clamped monolayers of Caco-2 epithelia, mounted in Ussing chambers, were exposed to apical substrate in Na(+)-free media, with apical pH held at 6.0. The D-cycloserine dependent increase in Isc was dose-dependent with an apparent Km = 15.8 +/- 2.0 (mean +/- s.e. mean) mM, and Vmax = 373 +/- 21 nmol cm-2h-1. 4. D-Cycloserine (20 mM) induced a prompt acidification of Caco-2 cell cytosol when superfused at the apical surface in both Na+ and Na(+)-free conditions. Cytosolic acidification in response to D-cycloserine was dependent upon superfusate pH, being attenuated at pH 8 and enhanced in acidic media. 5. The increment in Isc with 20 mM D-cycloserine was non-additive with other amino acid substrates for H+/amino acid symport.

Production of the amino acid 2-aminobutyrate was studied in four strains of Megasphaera elsdenii grown on a lactate-based growth medium containing Bacto-casamino acids and yeast extract. Supplementation with threonine increased the production of 2-aminobutyrate in three of the four strains, but no substantial increase in production was noted with serine, methionine, or aspartate, all of which are potential sources for the precursor of 2-aminobutyrate, 2-oxobutyrate. L-Cycloserine, an inhibitor of alanine transaminases, decreased both alanine and 2-aminobutyrate production, suggesting that 2-aminobutyrate synthesis may share the same metabolic pathway as alanine synthesis or that 2-oxobutyrate can act as a substrate for alanine transaminases. Decreases in the production of 2-aminobutyrate were associated with a reduction in the catabolism of branched-chain amino acids in two of the four strains.

The kinetic mechanism for the interaction of D-cycloserine with serine hydroxymethyltransferase (EC 2.1.2.1) from sheep liver was established by measuring changes in the activity, absorbance, and circular dichoism (CD) of the enzyme. The irreversible inhibition of the enzyme was characterized by three detectable steps: an initial rapid step followed by two successive steps with rate constants of 5.4 X 10(-3) s-1 and 1.4 X 10(-4) s-1. The first step was distinguished by a rapid disappearance of the enzyme absorbance peak at 425 nm, a decrease in the enzyme activity to 25% of the uninhibited velocity, and a lowering of the CD intensity at 432 nm to about 65% of the original value. The second step of the interaction was accompanied by a complete loss of enzyme activity and a marginal increase in the CD intensity at 432 nm. The final step resulted in the complete loss of the enzyme absorbance at 425 nm and of the CD band at 432 nm. The products of the reaction were identified as (a) apoenzyme by absorbance measurements, CD spectra, and reconstitution with pyridoxal 5'-phosphate and (b) a pyridoxal 5'-phosphate-D-cycloserine Schiff's base complex identified by its fluorescence and absorbance spectra. The Schiff base complex was expelled from the enzyme active site in the final step of the reaction. The proposed mechanism, which is different from those operative in other pyridoxal phosphate dependent enzymes, probably accounts for the selective inhibition of serine hydroxymethyltransferase by the drug in vivo.

The uptake of antibiotics into bacterial cells depends on the structures and mechanisms involved in general transport of substrates. Because of their different molecular architecture, the distinct layers of the cell envelope differ in permeability. The cell-wall of gram-positive bacteria, which is simply structured, is therefore more permeable to a series of antibiotics than is the gram-negative cell-wall which is built like other biological membranes. The energy dependent uptake of many antimetabolic antibiotics in managed by the selective transport systems of the cytoplasmic membrane. In the case of antibiotics, which are not analogous in structure with metabolites, the dependence of the drug on active transport is less obvious. The uptake of antibiotics may even be influenced by the target-site of the drug in the cytoplasm. An example is the accumulation of aminoglycoside antibiotics which is initiated by binding of the drugs to the ribosome. The autoinduction of aminoglycoside uptake is associated with an increased transport of polyamines. The broad variety of modes of resistance, based on permeability, results from the large number of components involved in the uptake of antibiotics. Resistance of bacteria can depend on alterations of every layer of the cell envelope. The existence of a capsule can cause resistance, as well as the specific loss of protein components of the outer membrane, which are involved in the uptake of special substrates. The exclusion of antimetabolites by the loss of the cognate transport system yields well defined resistance. By contrast, resistance towards a wide variety of unrelated antibiotics results from the uncoupling of energization and transport phenomena. Resistance based on reduced uptake becomes even more complex when the modification of the target site alters the accumulation of antibiotics. Far beyond common mechanisms is the ability of cells to actively excrete antibiotics. Far beyond common mechanisms is the ability of cells to actively excrete antibiotics that have already penetrated. The induction of selective export systems for tetracyclines is not dependent on activities of cell-borne transport systems but is a consequence of the acquisition of resistance plasmids.

Resistance to d-cycloserine could be induced in Bacillus subtilis 168 by sublethal concentrations of d-cycloserine. Sensitivity to the antibiotic could be regained by growth in the absence of d-cycloserine. The bactericidal activity of d-cycloserine apparently was not altered by resistant cells, and peptidoglycan synthesis was still inhibited by d-cycloserine in resistant cells. The d-cycloserine resistance apparently resulted from a decreased uptake of the antibiotic. The decrease in d-cycloserine transport could be prevented by simultaneous treatment of the cells with rifampin and d-cycloserine. d-Cycloserine was transported by the same system as glycine in B. subtilis. d-Cycloserine was able to exchange for intracellular glycine in both sensitive and resistant cells, suggesting that d-cycloserine is not excluded from the cell in resistant cultures.

The influence of bile flow on the absorption of tetracycline, doxycycline, sulphadimidine and cycloserine was studied in 13 volunteer patients with t-tubes in their main bile ducts after biliary surgery. Their hepatic functions and bile flows were estimated by giving I131 radioiodinated rose-bengal intravenously. About 80% radioactive tracer was recovered from the bile when the t-tube was open, so most of the bile was diverted outside the intestinal tract. Up to 4 hours after administration serum tetracycline levels were lower when the t-tube was open than when the t-tube was closed. In one patient the serum levels were so much reduced that therapeutic serum tetracycline levels could not be achieved. The serum doxycycline levels were fairly high and bile flow did not have any effect on absorption. The 24-hour biliary excretion of doxycycline was only about 15% of the urinary excretion. The absorption of sulphadimidine and cycloserine was not affected by the presence or absence of bile. The bile salts are important surfactants in man, and modify the absorption rate of tetracycline, but not of doxycycline, sulphadimidine and cycloserine. Even in the absence of intact bile flow therapeutic serum tetracycline levels can be expected with the doses currently used.

A short historical review and pharmacological data indicating the suitability of cycloserine for long-term low-dose prophylaxis of urinary tract infection are presented.

Whole cells of Bacillus subtilis transported d-alanine and l-alanine by two different systems. The high-affinity system (K(m) of 1 muM and V(max) of 0.6 to 0.8 nmol/min per mg of protein) was specific for the two stereoisomers of alanine. The low-affinity system (K(m) of 10 muM for l-alanine and 20 muM for d-alanine and glycine) had a V(max) of 5 to 12 nmol/min per mg of protein. This system transported glycine, d-cycloserine, and d-serine, in addition to d- and l-alanine. Azide inhibited the uptake of these amino acids and caused the efflux of d-alanine from preloaded cells. These data suggest that transport of these amino acids is energized by the electron transport chain.

A quantitative study of the contribution of the malate-aspartate and glycerol-3-phosphate cycles to the translocation of reducing equivalents from cytosol to mitochondria during ethanol oxidation has been made in hepatocytes from euthyroid and hyperthyroid rats. 1. In hepatocytes from euthyroid rats both cycles have an almost equal capacity and their relative contribution to total hydrogen transport to the mitochondria depends on the conditions chosen. 2. In hepatocytes from hyperthyroid rats maximal rates of ethanol oxidation were significantly lower than in hepatocytes from euthyroid rats, even though the capacity of the glycerol-3-phosphate cycle was increased. This was due to a decreased activity of alcohol dehydrogenase.

We present evidence which demonstrates that L-cycloserine, structural analog of L-alanine, which is known to be an effective aminotransferase inhibitor, is also a potent inhibitor of cellular pyruvate metabolism. This effect was found to be related to its almost instantaneous action in decreasing pyruvate concentrations in a dose-dependent manner. 1H nuclear magnetic resonance studies clearly demonstrate that the irreversible removal of pyruvate induced by L-cycloserine is caused by the decarboxylating action of the latter. Pyruvate disappearance induced by L-cycloserine can be stoichiometrically accounted for as acetate. The process does not involve any chemically detected transformation of L-cycloserine. These observations lead to two main considerations regarding the known action of L-cycloserine. First, its inhibitory effect on gluconeogenesis from lactate could be explained only on the basis of its ability to reduce pyruvate availability with no apparent need for transaminase inhibition. Second, its ability as a transaminase inhibitor should be reconsidered in view of its potent decarboxylating action on pyruvate and probably other oxoacids.