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Biotin synthesis begins by hijacking the fatty acid synthetic pathway.

Lin S, Hanson RE, Cronan JE - Nat. Chem. Biol. (2010)

Bottom Line: Although biotin is an essential enzyme cofactor found in all three domains of life, our knowledge of its biosynthesis remains fragmentary.We report in vivo and in vitro evidence that the pimeloyl moiety is synthesized by a modified fatty acid synthetic pathway in which the omega-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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Illinois, Urbana, Illinois, USA.

ABSTRACT
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 this intermediate is assembled remains unknown. Genetic analysis in Escherichia coli 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 omega-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.

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Inhibition of DTB synthesis by inhibitors of fatty acid synthesis and SAM-dependent methyltransferasesThe microbiocide, triclosan, or the antibiotics cerulenin, thiolactomycin and sinefungin were added to the in vitro DTB synthesis with either malonyl-CoA (○) or pimeloyl-ACP methyl ester (■) as substrate. The level of DTB produced was estimated based on the area of the bioassay red zone and was standardized to the control reactions containing no inhibitor (100 % DTB synthesis was approximately 30 ρmol). The lack of inhibition seen when pimeloyl-ACP methyl ester was the substrate demonstrated that the amounts of inhibitor transferred to the assay disks were insufficient to inhibit growth of the bioassay strain ER90 (ΔbioF bioC bioD). Inhibition by triclosan was reduced when FabV, a triclosan resistant enoyl-ACP reductase, was added to 50 µg/ml in the reaction (●). The protein synthesis inhibitor, kanamycin, was tested with malonyl-CoA as the substrate and gave no inhibition over the same range of concentrations. A bioassay plate showing the effects of thiolactomycin on DTB synthesis is given at the lower right of the figure. Type of file: figure
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Figure 4: Inhibition of DTB synthesis by inhibitors of fatty acid synthesis and SAM-dependent methyltransferasesThe microbiocide, triclosan, or the antibiotics cerulenin, thiolactomycin and sinefungin were added to the in vitro DTB synthesis with either malonyl-CoA (○) or pimeloyl-ACP methyl ester (■) as substrate. The level of DTB produced was estimated based on the area of the bioassay red zone and was standardized to the control reactions containing no inhibitor (100 % DTB synthesis was approximately 30 ρmol). The lack of inhibition seen when pimeloyl-ACP methyl ester was the substrate demonstrated that the amounts of inhibitor transferred to the assay disks were insufficient to inhibit growth of the bioassay strain ER90 (ΔbioF bioC bioD). Inhibition by triclosan was reduced when FabV, a triclosan resistant enoyl-ACP reductase, was added to 50 µg/ml in the reaction (●). The protein synthesis inhibitor, kanamycin, was tested with malonyl-CoA as the substrate and gave no inhibition over the same range of concentrations. A bioassay plate showing the effects of thiolactomycin on DTB synthesis is given at the lower right of the figure. Type of file: figure

Mentions: The proposed pimelate synthetic pathway was further tested by use of the antibiotics, cerulenin and thiolactomycin plus the microbiocide, triclosan, to block fatty acid synthesis in vitro (Fig. 4). Cerulenin inhibits the β-ketoacyl-ACP synthases, FabB and FabF, whereas thiolactomycin inhibits FabB and FabF plus the short chain β-ketoacyl-ACP synthase, FabH23. Triclosan inhibits FabI, the sole E. coli enoyl-ACP reductase23. Addition of any of these three inhibitors severely reduced the levels of DTB synthesized from malonyl-CoA in a concentration-dependent manner (Fig. 4). Moreover, the effects of triclosan were shown to be due to inhibition of FabI because addition of purified Vibrio cholerae FabV, a naturally triclosan-resistant enoyl-ACP reductase24, largely restored DTB synthesis to triclosan-treated extracts. The fatty acid synthesis inhibitors had no effect on DTB synthesis when pimeloyl-ACP methyl ester was the substrate indicating that their effects were due to inhibition of synthesis of the pimeloyl moiety.


Biotin synthesis begins by hijacking the fatty acid synthetic pathway.

Lin S, Hanson RE, Cronan JE - Nat. Chem. Biol. (2010)

Inhibition of DTB synthesis by inhibitors of fatty acid synthesis and SAM-dependent methyltransferasesThe microbiocide, triclosan, or the antibiotics cerulenin, thiolactomycin and sinefungin were added to the in vitro DTB synthesis with either malonyl-CoA (○) or pimeloyl-ACP methyl ester (■) as substrate. The level of DTB produced was estimated based on the area of the bioassay red zone and was standardized to the control reactions containing no inhibitor (100 % DTB synthesis was approximately 30 ρmol). The lack of inhibition seen when pimeloyl-ACP methyl ester was the substrate demonstrated that the amounts of inhibitor transferred to the assay disks were insufficient to inhibit growth of the bioassay strain ER90 (ΔbioF bioC bioD). Inhibition by triclosan was reduced when FabV, a triclosan resistant enoyl-ACP reductase, was added to 50 µg/ml in the reaction (●). The protein synthesis inhibitor, kanamycin, was tested with malonyl-CoA as the substrate and gave no inhibition over the same range of concentrations. A bioassay plate showing the effects of thiolactomycin on DTB synthesis is given at the lower right of the figure. Type of file: figure
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2925990&req=5

Figure 4: Inhibition of DTB synthesis by inhibitors of fatty acid synthesis and SAM-dependent methyltransferasesThe microbiocide, triclosan, or the antibiotics cerulenin, thiolactomycin and sinefungin were added to the in vitro DTB synthesis with either malonyl-CoA (○) or pimeloyl-ACP methyl ester (■) as substrate. The level of DTB produced was estimated based on the area of the bioassay red zone and was standardized to the control reactions containing no inhibitor (100 % DTB synthesis was approximately 30 ρmol). The lack of inhibition seen when pimeloyl-ACP methyl ester was the substrate demonstrated that the amounts of inhibitor transferred to the assay disks were insufficient to inhibit growth of the bioassay strain ER90 (ΔbioF bioC bioD). Inhibition by triclosan was reduced when FabV, a triclosan resistant enoyl-ACP reductase, was added to 50 µg/ml in the reaction (●). The protein synthesis inhibitor, kanamycin, was tested with malonyl-CoA as the substrate and gave no inhibition over the same range of concentrations. A bioassay plate showing the effects of thiolactomycin on DTB synthesis is given at the lower right of the figure. Type of file: figure
Mentions: The proposed pimelate synthetic pathway was further tested by use of the antibiotics, cerulenin and thiolactomycin plus the microbiocide, triclosan, to block fatty acid synthesis in vitro (Fig. 4). Cerulenin inhibits the β-ketoacyl-ACP synthases, FabB and FabF, whereas thiolactomycin inhibits FabB and FabF plus the short chain β-ketoacyl-ACP synthase, FabH23. Triclosan inhibits FabI, the sole E. coli enoyl-ACP reductase23. Addition of any of these three inhibitors severely reduced the levels of DTB synthesized from malonyl-CoA in a concentration-dependent manner (Fig. 4). Moreover, the effects of triclosan were shown to be due to inhibition of FabI because addition of purified Vibrio cholerae FabV, a naturally triclosan-resistant enoyl-ACP reductase24, largely restored DTB synthesis to triclosan-treated extracts. The fatty acid synthesis inhibitors had no effect on DTB synthesis when pimeloyl-ACP methyl ester was the substrate indicating that their effects were due to inhibition of synthesis of the pimeloyl moiety.

Bottom Line: Although biotin is an essential enzyme cofactor found in all three domains of life, our knowledge of its biosynthesis remains fragmentary.We report in vivo and in vitro evidence that the pimeloyl moiety is synthesized by a modified fatty acid synthetic pathway in which the omega-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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Illinois, Urbana, Illinois, USA.

ABSTRACT
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 this intermediate is assembled remains unknown. Genetic analysis in Escherichia coli 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 omega-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.

Show MeSH
Related in: MedlinePlus