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Studies on the biosynthesis of biotin. Production of biotin and biotin-like compounds by a pseudomonad.

ABSTRACT: 1. Filtrates from cultures of a strain of Pseudomonas aeruginosa, grown in a basal glucose-ammonium chloride-vitamins-salts medium, possessed biotin activity as detected by microbiological assays. Exponential-phase culture filtrates contained biotin and desthiobiotin in the approximate ratio 1:3, with smaller amounts of biotin sulphoxide and three unidentified compounds with biotin activity. 2. The addition of malonate, adipate or pimelate to the basal medium stimulated the production of compounds with biotin activity; this effect was enhanced when these compounds were included in the medium as the major carbon source. Succinate, glutarate, suberate, fumarate or oxaloacetate did not stimulate the production of compounds with biotin activity. The ratio of biotin to desthiobiotin in filtrates from cultures grown in medium containing malonate as the carbon source was about 1:1. Experiments in which mixtures of malonate and pimelate were included in the medium as the carbon sources showed that these acids probably make a similar contribution in biotin biosynthesis. 3. A number of heterocyclic compounds, including several containing the ureido group (-NH-CO-NH-), were included in the basal medium but none of them stimulated the production of compounds with biotin activity to any marked degree. 4. Several amino acids, particularly cysteine (or cystine) and lysine, when added individually as supplements to the basal medium, stimulated the production of compounds with biotin activity. Filtrates from cultures grown in medium supplemented with cysteine contained approximately equal proportions of biotin and desthiobiotin. A much greater stimulation in the production of compounds with biotin activity was obtained when certain amino acids were included in the medium as the major source of nitrogen or carbon and nitrogen; ornithine, citrulline and argininosuccinate had the most marked effect. The ratio of biotin to desthiobiotin in filtrates from these cultures was usually greater than in filtrates from cultures grown in basal medium. 5-Aminovalerate also caused some stimulation when used as the nitrogen source, but urea was inactive. The effect of binary mixtures of certain amino acids was also examined. 5. The results are discussed in relation to the possible role of the stimulatory compounds during biotin biosynthesis.

SUBMITTER: Rose AH

PROVIDER: S-EPMC1207042 | biostudies-other | 1965 Aug

REPOSITORIES: biostudies-other

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Project description:Transcription of the Escherichia coli biotin (bio) operon is directly regulated by the biotin protein ligase BirA, the enzyme that covalently attaches biotin to its cognate acceptor proteins. Binding of BirA to the bio operator requires dimerization of the protein, which is triggered by BirA-catalyzed synthesis of biotinoyl-adenylate (biotinoyl-5'-AMP), the obligatory intermediate of the ligation reaction. Although several aspects of this regulatory system are well understood, no BirA superrepressor mutant strains had been isolated. Such superrepressor BirA proteins would repress the biotin operon transcription in vivo at biotin concentrations well below those needed for repression by wild-type BirA. We isolated mutant strains having this phenotype by a combined selection-screening approach and resolved multiple mutations to give several birA superrepressor alleles, each having a single mutation, all of which showed repression dominant over that of the wild-type allele. All of these mutant strains repressed bio operon transcription in vivo at biotin concentrations that gave derepression of the wild-type strain and retained sufficient ligation activity for growth when overexpressed. All of the strains except that encoding G154D BirA showed derepression of bio operon transcription upon overproduction of a biotin-accepting protein. In BirA, G154D was a lethal mutation in single copy, and the purified protein was unable to transfer biotin from enzyme-bound biotinoyl-adenylate either to the natural acceptor protein or to a biotin-accepting peptide sequence. Consistent with the transcriptional repression data, each of the purified mutant proteins showed increased affinity for the biotin operator DNA in electrophoretic mobility shift assays. Surprisingly, although most of the mutations were located in the catalytic domain, all of those tested, except G154D BirA, had normal ligase activity. Most of the mutations that gave superrepressor phenotypes altered residues located close to the dimerization interface of BirA. However, two mutations were located at sites well removed from the interface. The properties of the superrepressor mutants strengthen and extend other data indicating that BirA function entails extensive interactions among the three domains of the protein and show that normal ligase activity does not ensure normal DNA binding.

Project description:Pimeloyl-acyl carrier protein (ACP) methyl ester esterase catalyzes the last biosynthetic step of the pimelate moiety of biotin, a key intermediate in biotin biosynthesis. The paradigm pimeloyl-ACP methyl ester esterase is the BioH protein of Escherichia coli that hydrolyses the ester bond of pimeloyl-ACP methyl ester. Biotin synthesis in E. coli also requires the function of the malonyl-ACP methyltransferase gene (bioC) to employ a methylation strategy to allow elongation of a temporarily disguised malonate moiety to a pimelate moiety by the fatty acid synthetic enzymes. However, bioinformatics analyses of the extant bacterial genomes showed that bioH is absent in many bioC-containing bacteria. The genome of the Gram-negative bacterium, Moraxella catarrhalis lacks a gene encoding a homolog of any of the six known pimeloyl-ACP methyl ester esterase isozymes suggesting that this organism encodes a novel pimeloyl-ACP methyl ester esterase isoform. We report that this is the case. The gene encoding the new isoform, called btsA, was isolated by complementation of an E. coli bioH deletion strain. The requirement of BtsA for the biotin biosynthesis in M. catarrhalis was confirmed by a biotin auxotrophic phenotype caused by deletion of btsA in vivo and a reconstituted in vitro desthiobiotin synthesis system. Purified BtsA was shown to cleave the physiological substrate pimeloyl-ACP methyl ester to pimeloyl-ACP by use of a Ser117-His254-Asp287 catalytic triad. The lack of sequence alignment with other isozymes together with phylogenetic analyses revealed BtsA as a new class of pimeloyl-ACP methyl ester esterase. The involvement of BtsA in M. catarrhalis virulence was confirmed by the defect of bacterial invasion to lung epithelial cells and survival within macrophages in the ΔbtsA strains. Identification of the new esterase gene btsA exclusive in Moraxella species that links biotin biosynthesis to bacterial virulence, can reveal a new valuable target for development of drugs against M. catarrhalis.

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Studies on the biosynthesis of biotin. Production of biotin and biotin-like compounds by a pseudomonad.

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