Ed to another moiety. A thioester seems the most likely linkage (14, 15). It should be noted that biotin is required for synthesis of malonyl-CoA, the postulated source of all of the carbon atoms of the pimelate moiety. Hence, we are presented with an evolutionary conundrum, biotin is required for biotin synthesis.Author Manuscript Author Manuscript Author Manuscript Author ManuscriptThe Genes of Biotin SynthesisBiotin requiring mutants of E. coli were first isolated many years ago. All of the mutants isolated as biotin auxotrophs were clustered at min 17 of the genetic map and defined five genes, called bioABCDF, based on mapping, cross-feeding and complementation studies (16?8). However, during deletion analysis of the maltose utilization genes, a strain that required biotin was isolated and called bioH (19). More recently, strains having a nonfunctional pfs (now called mtn), the gene encoding 5-methylthioadenosine/Sadenosylhomocysteine nucleosidase were shown to require biotin due to accumulation of an inhibitory metabolite (see below) (20, 21). The fact that biotin auxotrophs due to mutations in the bioH or mtn genes were not isolated by classical means is probably due to the fact that E. coli biotin auxotrophs require only miniscule amounts of this cofactor (supplementation with biotin at concentrations of a few nanomolar) and that conventional undefined growth media are often very rich in biotin. Therefore, often several platings on biotin-free media are required to detect the biotin requirement of auxotrophs.The Pathway and Proteins of Biotin SynthesisThe late steps of biotin biosynthesis (Fig. 2) were worked out many years ago whereas the early steps have only recently been elucidated. The proteins (BioA, BioB, BioD and BioF) of the late steps are well-studied enzymes of known crystal structures whereas the proteins of the early steps, BioC and BioH, were much more poorly understood. The sequence of steps in the late pathway was readily deduced since E. coli takes up each of the lateEcoSal Plus. Author manuscript; available in PMC 2015 January 06.CronanPageintermediates. Growth of mutants with lesions in bioC and bioH Duvoglustat site proceeds when the medium is supplemented with 7-keto-8-amino pelargonic acid (KAPA) or any of the later intermediates in the pathway. No cross-feeding is observed between bioC and bioH mutant strains suggesting that the early intermediates may not pass through cell membranes, perhaps because they are Leupeptin (hemisulfate) manufacturer protein-bound. In recent years the functions of BioC and BioH has become clear more then 50 years after the genes were discovered. The question was how to assemble a seven-carbon dicarboxylic acid in E. coli. BioC was annotated as an S-adenosylL-methionine (SAM)-dependent methyltransferase whereas BioH had been shown to have esterase activity on a series of short and medium chain acyl p-nitrophenyl esters(13, 22, 23) and on the methyl ester of dimethylbutyryl-S-methyl mercaptopropionate (24). The BioC annotation was especially puzzling because biotin contains no methyl groups and (as discussed above) all of the pimeloyl moiety carbon atoms are derived from acetate, alanine and CO2. Thus, it seemed that assembly of the pimeloyl moiety must require enzymes of another biosynthetic pathway that are somehow assisted in this task by BioC and BioH. Many years ago Lynen and coworkers (25) suggested that pimeloyl-CoA could be formed by the enzymes of fatty acid synthesis. They proposed that three molecules of malonyl-CoA would.Ed to another moiety. A thioester seems the most likely linkage (14, 15). It should be noted that biotin is required for synthesis of malonyl-CoA, the postulated source of all of the carbon atoms of the pimelate moiety. Hence, we are presented with an evolutionary conundrum, biotin is required for biotin synthesis.Author Manuscript Author Manuscript Author Manuscript Author ManuscriptThe Genes of Biotin SynthesisBiotin requiring mutants of E. coli were first isolated many years ago. All of the mutants isolated as biotin auxotrophs were clustered at min 17 of the genetic map and defined five genes, called bioABCDF, based on mapping, cross-feeding and complementation studies (16?8). However, during deletion analysis of the maltose utilization genes, a strain that required biotin was isolated and called bioH (19). More recently, strains having a nonfunctional pfs (now called mtn), the gene encoding 5-methylthioadenosine/Sadenosylhomocysteine nucleosidase were shown to require biotin due to accumulation of an inhibitory metabolite (see below) (20, 21). The fact that biotin auxotrophs due to mutations in the bioH or mtn genes were not isolated by classical means is probably due to the fact that E. coli biotin auxotrophs require only miniscule amounts of this cofactor (supplementation with biotin at concentrations of a few nanomolar) and that conventional undefined growth media are often very rich in biotin. Therefore, often several platings on biotin-free media are required to detect the biotin requirement of auxotrophs.The Pathway and Proteins of Biotin SynthesisThe late steps of biotin biosynthesis (Fig. 2) were worked out many years ago whereas the early steps have only recently been elucidated. The proteins (BioA, BioB, BioD and BioF) of the late steps are well-studied enzymes of known crystal structures whereas the proteins of the early steps, BioC and BioH, were much more poorly understood. The sequence of steps in the late pathway was readily deduced since E. coli takes up each of the lateEcoSal Plus. Author manuscript; available in PMC 2015 January 06.CronanPageintermediates. Growth of mutants with lesions in bioC and bioH proceeds when the medium is supplemented with 7-keto-8-amino pelargonic acid (KAPA) or any of the later intermediates in the pathway. No cross-feeding is observed between bioC and bioH mutant strains suggesting that the early intermediates may not pass through cell membranes, perhaps because they are protein-bound. In recent years the functions of BioC and BioH has become clear more then 50 years after the genes were discovered. The question was how to assemble a seven-carbon dicarboxylic acid in E. coli. BioC was annotated as an S-adenosylL-methionine (SAM)-dependent methyltransferase whereas BioH had been shown to have esterase activity on a series of short and medium chain acyl p-nitrophenyl esters(13, 22, 23) and on the methyl ester of dimethylbutyryl-S-methyl mercaptopropionate (24). The BioC annotation was especially puzzling because biotin contains no methyl groups and (as discussed above) all of the pimeloyl moiety carbon atoms are derived from acetate, alanine and CO2. Thus, it seemed that assembly of the pimeloyl moiety must require enzymes of another biosynthetic pathway that are somehow assisted in this task by BioC and BioH. Many years ago Lynen and coworkers (25) suggested that pimeloyl-CoA could be formed by the enzymes of fatty acid synthesis. They proposed that three molecules of malonyl-CoA would.
