The Experts below are selected from a list of 228 Experts worldwide ranked by ideXlab platform
Raul G Barletta - One of the best experts on this subject based on the ideXlab platform.
-
roles of mycobacterium smegmatis d alanine d alanine ligase and d alanine racemase in the mechanisms of action of and resistance to the peptidoglycan inhibitor d cycloserine
Antimicrobial Agents and Chemotherapy, 2003Co-Authors: Zhengyu Feng, Raul G BarlettaAbstract:d-Cycloserine (DCS) targets the peptidoglycan biosynthetic enzymes D-Alanine racemase (Alr) and D-Alanine:D-Alanine ligase (Ddl). Previously, we demonstrated that the overproduction of Alr in Mycobacterium smegmatis determines a DCS resistance phenotype. In this study, we investigated the roles of both Alr and Ddl in the mechanisms of action of and resistance to DCS in M. smegmatis. We found that the overexpression of either the M. smegmatis or the Mycobacterium tuberculosis ddl gene in M. smegmatis confers resistance to DCS, but at lower levels than the overexpression of the alr gene. Furthermore, a strain overexpressing both the alr and ddl genes displayed an eightfold-higher level of resistance. To test the hypothesis that inhibition of Alr by DCS decreases the intracellular pool of D-Alanine, we determined the alanine pools in M. smegmatis wild-type and recombinant strains with or without DCS treatment. Alr-overproducing strain GPM14 cells not exposed to DCS displayed almost equimolar amounts of l- and D-Alanine in the steady state. The wild-type strain and Ddl-overproducing strains contained a twofold excess of l- over D-Alanine. In all strains, DCS treatment led to a significant accumulation of l-alanine and a concomitant decease of D-Alanine, with approximately a 20-fold excess of l-alanine in the Ddl-overproducing strains. These data suggest that Ddl is not significantly inhibited by DCS at concentrations that inhibit Alr. This study is of significance for the identification of the lethal target(s) of DCS and the development of novel drugs targeting the D-Alanine branch of mycobacterial peptidoglycan biosynthesis.
-
mycobacterium smegmatis d alanine racemase mutants are not dependent on d alanine for growth
Antimicrobial Agents and Chemotherapy, 2002Co-Authors: Ofelia Chacon, Zhengyu Feng, Beth N Harris, Nancy E Caceres, Garry L Adams, Raul G BarlettaAbstract:Mycobacterium smegmatis is a fast-growing nonpathogenic species particularly useful in studying basic cellular processes of relevance to pathogenic mycobacteria. This study focused on the D-Alanine racemase gene (alrA), which is involved in the synthesis of D-Alanine, a basic component of peptidoglycan that forms the backbone of the cell wall. M. smegmatis alrA null mutants were generated by homologous recombination using a kanamycin resistance marker for insertional inactivation. Mutants were selected on Middlebrook medium supplemented with 50 mM D-Alanine and 20 μg of kanamycin per ml. These mutants were also able to grow in standard and minimal media without D-Alanine, giving rise to colonies with a drier appearance and more-raised borders than the wild-type strain. The viability of the mutants and independence of D-Alanine for growth indicate that inactivation of alrA does not impose an auxotrophic requirement for D-Alanine, suggesting the existence of a new pathway of D-Alanine biosynthesis in M. smegmatis. Biochemical analysis demonstrated the absence of any detectable D-Alanine racemase activity in the mutant strains. In addition, the alrA mutants displayed hypersusceptibility to the antimycobacterial agent d-cycloserine. The MIC of d-cycloserine for the mutant strain was 2.56 μg/ml, 30-fold less than that for the wild-type strain. Furthermore, this hypersusceptibility was confirmed by the bactericidal action of d-cycloserine on broth cultures. The kinetic of killing for the mutant strain followed the same pattern as that for the wild-type strain, but at a 30-fold-lower drug concentration. This effect does not involve a change in the permeability of the cell wall by this drug and is consistent with the identification of D-Alanine racemase as a target of d-cycloserine. This outcome is of importance for the design of novel antituberculosis drugs targeting peptidoglycan biosynthesis in mycobacteria.
Bengt Soderstrom - One of the best experts on this subject based on the ideXlab platform.
-
metabolism of n 15 alanine in the ectomycorrhizal fungus paxillus involutus
Experimental Mycology, 1995Co-Authors: Michel Chalot, Hans Ek, Roger D. Finlay, Bengt SoderstromAbstract:Alanine metabolism in the ectomycorrhizal fungus Paxillus involutus was investigated using [N-15]alanine. Short-term exposure of mycelial discs to [N-15]alanine showed that the greatest flow of N-15 was to glutamate and to aspartate. Levels of enrichment were as high as 15-20% for glutamate and 13-18% for aspartate, whereas that of alanine reached 30%. Label was also detected in the amino-N of glutamine and in serine and glycine, although at lower levels. Preincubation of mycelia with aminooxyacetate, an inhibitor of transamination reactions, resulted in complete inhibition of the flow of the label to glutamate, aspartate, and amino-N of glutamine, whereas [N-15]alanine rapidly accumulated. This evidence indicates the direct involvement of alanine aminotransferase for translocation of N-15 from alanine to glutamate. Alanine may be a convenient reservoir of both nitrogen and carbon. (C) 1995 Academic Press, Inc. (Less)
-
respiration of 14c alanine by the ectomycorrhizal fungus paxillus involutus
Fems Microbiology Letters, 1994Co-Authors: Michel Chalot, Annick Brun, Roger D. Finlay, Bengt SoderstromAbstract:The ectomycorrhizal fungus Paxillus involutus efficiently took up exogenously supplied [14C]alanine and rapidly converted it to pyruvate, citrate, succinate, fumarate and to CO2, thus providing direct evidence for the utilisation of alanine as a respiratory substrate. [14C]alanine was further actively metabolised to glutamate, glutamine and aspartate. Exposure to aminooxyacetate completely suppressed 14CO2 evolution and greatly reduced the flow of carbon from [14C]alanine to tricarboxylic acid cycle intermediates and amino acids, suggesting that alanine aminotransferase plays a pivotal role in alanine metabolism in Paxillus involutus.
Michel Chalot - One of the best experts on this subject based on the ideXlab platform.
-
metabolism of n 15 alanine in the ectomycorrhizal fungus paxillus involutus
Experimental Mycology, 1995Co-Authors: Michel Chalot, Hans Ek, Roger D. Finlay, Bengt SoderstromAbstract:Alanine metabolism in the ectomycorrhizal fungus Paxillus involutus was investigated using [N-15]alanine. Short-term exposure of mycelial discs to [N-15]alanine showed that the greatest flow of N-15 was to glutamate and to aspartate. Levels of enrichment were as high as 15-20% for glutamate and 13-18% for aspartate, whereas that of alanine reached 30%. Label was also detected in the amino-N of glutamine and in serine and glycine, although at lower levels. Preincubation of mycelia with aminooxyacetate, an inhibitor of transamination reactions, resulted in complete inhibition of the flow of the label to glutamate, aspartate, and amino-N of glutamine, whereas [N-15]alanine rapidly accumulated. This evidence indicates the direct involvement of alanine aminotransferase for translocation of N-15 from alanine to glutamate. Alanine may be a convenient reservoir of both nitrogen and carbon. (C) 1995 Academic Press, Inc. (Less)
-
respiration of 14c alanine by the ectomycorrhizal fungus paxillus involutus
Fems Microbiology Letters, 1994Co-Authors: Michel Chalot, Annick Brun, Roger D. Finlay, Bengt SoderstromAbstract:The ectomycorrhizal fungus Paxillus involutus efficiently took up exogenously supplied [14C]alanine and rapidly converted it to pyruvate, citrate, succinate, fumarate and to CO2, thus providing direct evidence for the utilisation of alanine as a respiratory substrate. [14C]alanine was further actively metabolised to glutamate, glutamine and aspartate. Exposure to aminooxyacetate completely suppressed 14CO2 evolution and greatly reduced the flow of carbon from [14C]alanine to tricarboxylic acid cycle intermediates and amino acids, suggesting that alanine aminotransferase plays a pivotal role in alanine metabolism in Paxillus involutus.
Zhengyu Feng - One of the best experts on this subject based on the ideXlab platform.
-
roles of mycobacterium smegmatis d alanine d alanine ligase and d alanine racemase in the mechanisms of action of and resistance to the peptidoglycan inhibitor d cycloserine
Antimicrobial Agents and Chemotherapy, 2003Co-Authors: Zhengyu Feng, Raul G BarlettaAbstract:d-Cycloserine (DCS) targets the peptidoglycan biosynthetic enzymes D-Alanine racemase (Alr) and D-Alanine:D-Alanine ligase (Ddl). Previously, we demonstrated that the overproduction of Alr in Mycobacterium smegmatis determines a DCS resistance phenotype. In this study, we investigated the roles of both Alr and Ddl in the mechanisms of action of and resistance to DCS in M. smegmatis. We found that the overexpression of either the M. smegmatis or the Mycobacterium tuberculosis ddl gene in M. smegmatis confers resistance to DCS, but at lower levels than the overexpression of the alr gene. Furthermore, a strain overexpressing both the alr and ddl genes displayed an eightfold-higher level of resistance. To test the hypothesis that inhibition of Alr by DCS decreases the intracellular pool of D-Alanine, we determined the alanine pools in M. smegmatis wild-type and recombinant strains with or without DCS treatment. Alr-overproducing strain GPM14 cells not exposed to DCS displayed almost equimolar amounts of l- and D-Alanine in the steady state. The wild-type strain and Ddl-overproducing strains contained a twofold excess of l- over D-Alanine. In all strains, DCS treatment led to a significant accumulation of l-alanine and a concomitant decease of D-Alanine, with approximately a 20-fold excess of l-alanine in the Ddl-overproducing strains. These data suggest that Ddl is not significantly inhibited by DCS at concentrations that inhibit Alr. This study is of significance for the identification of the lethal target(s) of DCS and the development of novel drugs targeting the D-Alanine branch of mycobacterial peptidoglycan biosynthesis.
-
mycobacterium smegmatis d alanine racemase mutants are not dependent on d alanine for growth
Antimicrobial Agents and Chemotherapy, 2002Co-Authors: Ofelia Chacon, Zhengyu Feng, Beth N Harris, Nancy E Caceres, Garry L Adams, Raul G BarlettaAbstract:Mycobacterium smegmatis is a fast-growing nonpathogenic species particularly useful in studying basic cellular processes of relevance to pathogenic mycobacteria. This study focused on the D-Alanine racemase gene (alrA), which is involved in the synthesis of D-Alanine, a basic component of peptidoglycan that forms the backbone of the cell wall. M. smegmatis alrA null mutants were generated by homologous recombination using a kanamycin resistance marker for insertional inactivation. Mutants were selected on Middlebrook medium supplemented with 50 mM D-Alanine and 20 μg of kanamycin per ml. These mutants were also able to grow in standard and minimal media without D-Alanine, giving rise to colonies with a drier appearance and more-raised borders than the wild-type strain. The viability of the mutants and independence of D-Alanine for growth indicate that inactivation of alrA does not impose an auxotrophic requirement for D-Alanine, suggesting the existence of a new pathway of D-Alanine biosynthesis in M. smegmatis. Biochemical analysis demonstrated the absence of any detectable D-Alanine racemase activity in the mutant strains. In addition, the alrA mutants displayed hypersusceptibility to the antimycobacterial agent d-cycloserine. The MIC of d-cycloserine for the mutant strain was 2.56 μg/ml, 30-fold less than that for the wild-type strain. Furthermore, this hypersusceptibility was confirmed by the bactericidal action of d-cycloserine on broth cultures. The kinetic of killing for the mutant strain followed the same pattern as that for the wild-type strain, but at a 30-fold-lower drug concentration. This effect does not involve a change in the permeability of the cell wall by this drug and is consistent with the identification of D-Alanine racemase as a target of d-cycloserine. This outcome is of importance for the design of novel antituberculosis drugs targeting peptidoglycan biosynthesis in mycobacteria.
Nobuyoshi Esaki - One of the best experts on this subject based on the ideXlab platform.
-
functional characterization of alanine racemase from schizosaccharomyces pombe a eucaryotic counterpart to bacterial alanine racemase
Journal of Bacteriology, 2001Co-Authors: Takuma Uo, Tohru Yoshimura, Naotaka Tanaka, Kaoru Takegawa, Nobuyoshi EsakiAbstract:D-Alanine is an essential component of bacterial peptidoglycans and is produced by alanine racemase (EC 5.1.1.1), a pyridoxal 5′-phosphate (PLP)-dependent enzyme (22, 29). However, D-Alanine also occurs in various natural compounds produced by other organisms. For example, cyclosporin A contains D-Alanine as a component and is produced by a fungus, Tolypocladium niveum (10). Alanine racemase was shown to be involved in the biosynthesis of D-Alanine in this fungus and was later purified and characterized. Even though it differs markedly in primary structure from bacterial alanine racemase, it rather resembles yeast threonine aldolase (4, 10). d-Serine occurs in mammalian brains and serves as an endogenous ligand of the glycine site of the N-methyl-d-aspartate receptor (14). Serine racemase, a PLP-dependent enzyme, was purified from rat brain to homogeneity (35), and a cDNA clone for mouse brain enzyme was obtained (36); this enzyme is distinct from both bacterial and fungal alanine racemases but similar to bacterial threonine dehydratase in primary structure (36). Serine racemase occurs also in vancomycin-resistant Enterococcus gallinarum (1). However, it resembles bacterial alanine racemases but not brain serine racemase. Therefore, alanine racemases and serine racemases are classified into three groups: bacterial alanine racemases plus serine racemase from E. gallinarum, fungal alanine racemase, and serine racemase of mammalian brain. Similar structural divergence has been found between other types of PLP enzymes, as exemplified by ornithine decarboxylases. Bacterial and eucaryotic ornithine decarboxylases differ markedly from each other, and the eucaryotic one shares the same protein fold with bacterial alanine racemase (9). Contrary to a long-standing belief, various d-amino acids, including D-Alanine and d-serine, have been found in yeasts in a peptide-bound form (15). Although no evidence has been obtained for the occurrence of a free form of d-amino acids, they are most probably produced in yeast cells upon proteolytic cleavage of the peptides. Therefore, it is reasonable to assume that amino acid racemases occur in yeasts. In fact, we have found that fission yeast, Schizosaccharomyces pombe, has a gene encoding a putative amino acid racemase similar to bacterial alanine racemases and the serine racemase of E. gallinarum. We have named the gene alr1+ because it shows alanine racemase activity upon cloning and expression in E. coli. We report here the gene cloning, purification, and characterization of the gene product, alanine racemase ALR1p. Furthermore, we show that the Alr1p protein is involved in the catabolism of D-Alanine in S. pombe, which we have confirmed through construction of a deletion mutation of the gene in S. pombe and heterologous expression of the gene in Saccharomyces cerevisiae.
