Heterologous production of the D-cycloserine intermediate O-acetyl-L-serine in a human type II pulmonary cell model.

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.

Crystal structure of O-ureidoserine racemase found in the d-cycloserine biosynthetic pathway.

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.

D-Cycloserine destruction by alanine racemase and the limit of irreversible inhibition.

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.

Differential effects of D-cycloserine and amantadine on motor behavior and D2/3 receptor binding in the nigrostriatal and mesolimbic system of the adult rat.

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 D2/3R binding in the brain of adult rats in relation to motor behavior. D2/3R binding was determined with small animal SPECT in baseline and after challenge with DCS (20 mg/kg) or AMA (40 mg/kg) with [123I]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 D2/3R 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 D2/3R 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 D2/3R binding after DCS reflect a reduction of available dopamine throughout the mesolimbocortical system. In contrast, the reductions of D2/3R 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.

Amount of Cycloserine Emanating from Terizidone Metabolism and Relationship with Hepatic Function in Patients with Drug-Resistant Tuberculosis.

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 (Cmax) and trough concentration (Cmin), 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 (Kaf), 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. Cmax (R2 = 22%, p = 0.003) and Cmin (R2 = 10.6%, p = 0.044) were significantly associated with increased CB concentration. Cmax was significantly associated with increased Kaf (R2 = 10.1%, p = 0.048), while high CLm/F was significantly associated with decreased AST/ALT (R2 = 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.

Inhibition of D-Ala:D-Ala ligase through a phosphorylated form of the antibiotic D-cycloserine.

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.

Studies on cyanobacterial protein PipY shed light on structure, potential functions, and vitamin B6 -dependent epilepsy.

The Synechococcus elongatus COG0325 gene pipY functionally interacts with the nitrogen regulatory gene pipX. As a first step toward a molecular understanding of such interactions, we characterized PipY. This 221-residue protein is monomeric and hosts pyridoxal phosphate (PLP), binding it with limited affinity and losing it upon incubation with D-cycloserine. PipY crystal structures with and without PLP reveal a single-domain monomer folded as the TIM barrel of type-III fold PLP enzymes, with PLP highly exposed, fitting a role for PipY in PLP homeostasis. The mobile PLP phosphate-anchoring C-terminal helix might act as a trigger for PLP exchange. Exploiting the universality of COG0325 functions, we used PipY in site-directed mutagenesis studies to shed light on disease causation by epilepsy-associated mutations in the human COG0325 gene PROSC.

High-Level Heterologous Production of D-Cycloserine by Escherichia coli.

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).

The structural and mutational analyses of O-ureido-L-serine synthase necessary for D-cycloserine biosynthesis.

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 ß-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.

Molecular basis underlying Mycobacterium tuberculosis D-cycloserine resistance. Is there a role for ubiquinone and menaquinone metabolic pathways?

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.

Involvement of alanine racemase in germination of Bacillus cereus spores lacking an intact exosporium.

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.

Association of a D-alanyl-D-alanine carboxypeptidase gene with the formation of aberrantly shaped cells during the induction of viable but nonculturable Vibrio parahaemolyticus.

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.

Evaluation of cycloserine-cefoxitin fructose agar (CCFA), CCFA with horse blood and taurocholate, and cycloserine-cefoxitin mannitol broth with taurocholate and lysozyme for recovery of Clostridium difficile isolates from fecal samples.

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.

Establishment of an in vitro D-cycloserine-synthesizing system by using O-ureido-L-serine synthase and D-cycloserine synthetase found in the biosynthetic pathway.

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.

Characterization of Escherichia coli D-cycloserine transport and resistant mutants.

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.

Structural and mutational studies on substrate specificity and catalysis of Salmonella typhimurium D-cysteine desulfhydrase.

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 ß-chloro-D-alanine (ßCDA). 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, ßCDA, 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 ßCDA 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.

Novel use of tryptose sulfite cycloserine egg yolk agar for isolation of Clostridium perfringens during an outbreak of necrotizing enterocolitis in a neonatal unit.

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.

Control of secondary metabolism by farX, which is involved in the gamma-butyrolactone biosynthesis of Streptomyces lavendulae FRI-5.

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.

D-cycloserine improves functional outcome after traumatic brain injury with wide therapeutic window.

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.