A Canonical Biotin Synthesis Enzyme, 8-Amino-7-Oxononanoate Synthase (BioF), Utilizes Different Acyl Chain Donors in Bacillus subtilis and Escherichia coli (original) (raw)
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Biochemical Journal, 1992
The pimeloyl-CoA synthase from Bacillus sphaericus has been purified to homogeneity from an overproducing strain of Escherichia coli. The purification yielded milligram quantities of the synthase with a specific activity of 1 unit/mg of protein. Analysis of the products showed that this enzyme catalysed the transformation of pimelate into pimeloyl-CoA with concomitant hydrolysis of ATP to AMP. Using a continuous spectrophotometric assay, we have examined the catalytic properties of the pure enzyme. The pH profile under Vmax. conditions showed a maximum around 8.5. Apparent Km values for pimelate, CoASH, ATP. Mg2- and Mg2+ were respectively 145 microM, 33 microM, 170 microM and 2.3 mM. The enzyme was inhibited by Mg2+ above 10 mM. This acid-CoA ligase exhibited a very sharp substrate specificity, e.g. neither GTP nor pimelate analogues (di- or mono-carboxylic acids) were processed. The bivalent metal ion requirement was also investigated: Mn2+ (73%) and Co2+ (32%) but not Ca2+ could ...
Nature Communications, 2020
Pimelic acid, a seven carbon α,ω-dicarboxylic acid (heptanedioic acid), is known to provide seven of the ten biotin carbon atoms including all those of the valeryl side chain. Distinct pimelate synthesis pathways were recently elucidated in Escherichia coli and Bacillus subtilis where fatty acid synthesis plus dedicated biotin enzymes produce the pimelate moiety. In contrast, the α-proteobacteria which include important plant and mammalian pathogens plus plant symbionts, lack all of the known pimelate synthesis genes and instead encode bioZ genes. Here we report a pathway in which BioZ proteins catalyze a 3-ketoacyl-acyl carrier protein (ACP) synthase III-like reaction to produce pimeloyl-ACP with five of the seven pimelate carbon atoms being derived from glutaryl-CoA, an intermediate in lysine degradation. Agrobacterium tumefaciens strains either deleted for bioZ or which encode a BioZ active site mutant are biotin auxotrophs, as are strains defective in CaiB which catalyzes glutar...
PLoS ONE, 2012
Biotin synthesis in Escherichia coli requires the functions of the bioH and bioC genes to synthesize the precursor pimelate moiety by use of a modified fatty acid biosynthesis pathway. However, it was previously noted that bioH has been replaced with bioG or bioK within the biotin synthetic gene clusters of other bacteria. We report that each of four BioG proteins from diverse bacteria and two cyanobacterial BioK proteins functionally replace E. coli BioH in vivo. Moreover, purified BioG proteins have esterase activity against pimeloyl-ACP methyl ester, the physiological substrate of BioH. Two of the BioG proteins block biotin synthesis when highly expressed and these toxic proteins were shown to have more promiscuous substrate specificities than the non-toxic BioG proteins. A postulated BioG-BioC fusion protein was shown to functionally replace both the BioH and BioC functions of E. coli. Although the BioH, BioG and BioK esterases catalyze a common reaction, the proteins are evolutionarily distinct.
Biotin synthesis begins by hijacking the fatty acid synthetic pathway
Nature Chemical Biology, 2010
Although biotin is an essential enzyme cofactor found in all three domains of life, our knowledge of its biosynthesis remains fragmentary. Most of the carbon atoms of biotin are derived from pimelic acid, a seven carbon dicarboxylic acid, but the mechanism whereby Escherichia coli assembles this intermediate remains unknown. Genetic analysis identified only two genes of unknown function required for pimelate synthesis, bioC and bioH. We report in vivo and in vitro evidence that the pimeloyl moiety is synthesized by a modified fatty acid synthetic pathway in which the ω-carboxyl group of a malonyl-thioester is methylated by BioC which allows recognition of this atypical substrate by the fatty acid synthetic enzymes. The malonyl-thioester methyl ester enters fatty acid synthesis as the primer and undergoes two reiterations of the fatty acid elongation cycle to give pimeloyl-acyl carrier protein (ACP) methyl ester which is hydrolyzed to pimeloyl-ACP and methanol by BioH. Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Current Opinion in Chemical Biology, 2011
Biotin synthesis requires the C7 α, ω-dicarboxylic acid, pimelic acid. Although pimelic acid was known to be primarily synthesized by a head to tail incorporation of acetate units, the synthetic mechanism was unknown. It has recently been demonstrated that in most bacteria the biotin pimelate moiety is synthesized by a modified fatty acid synthetic pathway in which the biotin synthetic intermediates are O-methyl esters disguised to resemble the canonical intermediates of the fatty acid synthetic pathway. Upon completion of the pimelate moiety, the methyl ester is cleaved. A very restricted set of bacteria have a different pathway in which the pimelate moiety is formed by cleavage of fatty acid synthetic intermediates by BioI, a member of the cytochrome P450 family.
Journal of Biological Chemistry, 2012
Background: The pimelate moiety of biotin is made by a modified fatty acid synthesis pathway. Results: The first reaction is O-methylation of the free carboxyl of malonyl-acyl carrier protein. Conclusion: The methyl acceptor is malonyl-acyl carrier protein and not malonyl-CoA. Significance: Demonstration of this enzymatic activity completes the E. coli biotin synthetic pathway. Recent work implicated the Escherichia coli BioC protein as the initiator of the synthetic pathway that forms the pimeloyl moiety of biotin (Lin, S., Hanson, R. E., and Cronan, J. E. (2010) Nat. Chem. Biol. 6, 682-688). BioC was believed to be an O-methyltransferase that methylated the free carboxyl of either malonyl-CoA or malonyl-acyl carrier protein based on the ability of O-methylated (but not unmethylated) precursors to bypass the BioC requirement for biotin synthesis both in vivo and in vitro. However, only indirect proof of the hypothesized enzymatic activity was obtained because the activities of the available BioC preparations were too low for direct enzymatic assay. Because E. coli BioC protein was extremely recalcitrant to purification in an active form, BioC homologues of other bacteria were tested. We report that the native form of Bacillus cereus ATCC10987 BioC functionally replaced E. coli BioC in vivo, and the protein could be expressed in soluble form and purified to homogeneity. In disagreement with prior scenarios that favored malonyl-CoA as the methyl acceptor, malonyl-acyl carrier protein was a far better acceptor of methyl groups from S-adenosyl-L-methionine than was malonyl-CoA. BioC was specific for the malonyl moiety and was inhibited by S-adenosyl-L-homocysteine and sinefungin. High level expression of B. cereus BioC in E. coli blocked cell growth and fatty acid synthesis. Biotin (vitamin H) is an essential enzyme cofactor required by all three domains of life (1, 2). It functions as a covalently bound prosthetic group that mediates the transfer of CO 2 in many vital metabolic carboxylation, decarboxylation, and transcarboxylation reactions (1, 2). Although biotin is essential, our knowledge of its biosynthesis remains fragmentary. 13 C-Labeling studies in Escherichia coli indicated that most of the carbon atoms of biotin are derived from pimelic acid, a seven-carbon ␣,-dicarboxylic acid (3, 4). However, only recently has the mechanism whereby E. coli assembles the pimeloyl intermediate been determined (5). Genetic studies in E. coli identified bioC and bioH as the only genes essential for biotin synthesis with unassigned functions (6). Strains having inactive bioC or bioH genes require biotin for growth, but biotin can be replaced by any of the later pathway intermediates including 7-keto-8aminopelargomic acid (formal name 8-amino-7-oxo-nonanoic acid) (6). Because 7-keto-8-aminopelargomic acid is readily synthesized in vitro from a thioester-linked pimeloyl moiety and L-alanine (6), BioC and BioH were assigned roles in pimelate synthesis. Various workers have proposed contradictory roles for BioC in biotin synthesis (7, 8). However, these proposals not only lacked supporting data but also failed to address the fundamental problem of how to assemble an ␣,-dicarboxylic acyl chain. Previously we reported the pathway of pimeloyl moiety synthesis in E. coli (5) (Fig. 1). In the pathway BioC and BioH do not directly catalyze the synthesis of pimelate but instead provide the means to allow fatty acid synthesis to assemble the pimelate moiety (Fig. 1). BioC catalyzes transfer of the methyl group of S-adenosyl-L-methionine (SAM) 2 to the-carboxyl group of malonyl-thioester of either CoA or acyl carrier protein (ACP) to form an O-methyl ester. The product then becomes the primer for the synthesis of pimeloyl-ACP by the fatty acid synthetic pathway. Methylation of the free carboxyl group of the malonyl-thioester was essential because of the extremely hydrophobic nature of the active sites of the fatty acid synthesis proteins (9). The methyl ester moiety neutralizes the carboxylate negative charge and mimics the methyl end of the monocarboxylates normally found in fatty acid synthesis. Two rounds of the standard fatty acid reductase-dehydratase-reductase reaction sequence results in the ACP thioester of-methyl pimelic acid (Fig. 1). At this stage the methyl group must be removed by BioH to block further elongation to azelayl-ACP methyl ester, a physiologically useless product (5). Moreover, the freed carboxyl group will later be required for the essential covalent attachment of biotin to its cognate enzymes (10). Although our prior work produced a pathway firmly based on both in vivo and in vitro data (5), a shortcoming was that our only source of BioC
Proceedings of the National Academy of Sciences, 2012
Although the pimeloyl moiety was long known to be a biotin precursor, the mechanism of assembly of this C7 α,ω-dicarboxylic acid was only recently elucidated. In Escherichia coli , pimelate is made by bypassing the strict specificity of the fatty acid synthetic pathway. BioC methylates the free carboxyl of a malonyl thioester, which replaces the usual acetyl thioester primer. This atypical primer is transformed to pimeloyl-acyl carrier protein (ACP) methyl ester by two cycles of fatty acid synthesis. The question is, what stops this product from undergoing further elongation? Although BioH readily cleaves this product in vitro, the enzyme is nonspecific, which made assignment of its physiological substrate problematical, especially because another enzyme, BioF, could also perform this gatekeeping function. We report the 2.05-Å resolution cocrystal structure of a complex of BioH with pimeloyl-ACP methyl ester and use the structure to demonstrate that BioH is the gatekeeper and its ph...
A Biotin Biosynthesis Gene Restricted to Helicobacter
Scientific reports, 2016
In most bacteria the last step in synthesis of the pimelate moiety of biotin is cleavage of the ester bond of pimeloyl-acyl carrier protein (ACP) methyl ester. The paradigm cleavage enzyme is Escherichia coli BioH which together with the BioC methyltransferase allows synthesis of the pimelate moiety by a modified fatty acid biosynthetic pathway. Analyses of the extant bacterial genomes showed that bioH is absent from many bioC-containing bacteria and is replaced by other genes. Helicobacter pylori lacks a gene encoding a homologue of the known pimeloyl-ACP methyl ester cleavage enzymes suggesting that it encodes a novel enzyme that cleaves this intermediate. We isolated the H. pylori gene encoding this enzyme, bioV, by complementation of an E. coli bioH deletion strain. Purified BioV cleaved the physiological substrate, pimeloyl-ACP methyl ester to pimeloyl-ACP by use of a catalytic triad, each member of which was essential for activity. The role of BioV in biotin biosynthesis was d...
The pimeloyl-CoA synthetase BioW defines a new fold for adenylate-forming enzymes
Nature Chemical Biology, 2017
Reactions that activate carboxylates through acyl-adenylate intermediates are found throughout biology and include acyl-and aryl-CoA synthetases and tRNA synthetases. Here we describe the characterization of Aquifex aeolicus BioW, which represents a new protein fold within the superfamily of adenylating enzymes. Substrate-bound structures identified the enzyme active site and elucidated the mechanistic strategy for conjugating CoA to the seven-carbon α,ωdicarboxylate pimelate, a biotin precursor. Proper position of reactive groups for the two halfreactions is achieved solely through movements of active site residues, as confirmed by sitedirected mutational analysis. The ability of BioW to hydrolyze adenylates of noncognate substrates is reminiscent of pre-transfer proofreading observed in some tRNA synthetases, and we show that this activity can be abolished by mutation of a single residue. These studies illustrate how BioW can carry out three different biologically prevalent chemical reactions (adenylation, thioesterification, and proofreading) in the context of a new protein fold. Biotin (vitamin B7 or coenzyme R) is a water-soluble essential cofactor in all domains of life, where it serves as the prosthetic group for numerous metabolic enzymes that catalyze Reprints and permissions information