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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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Related in: MedlinePlus

Requirements for DTB synthesis from malonyl-CoAThe cell free extract was prepared from the ΔbioC strain STL96 which carries multiple copies of all the bio operon genes except bioC. Purified and refolded BioC was added to all reactions except as shown. Bioassays were done using strain ER90 (ΔbioF) as described in Methods. (a) Standard DTB was bioassayed and reliably detected in levels as low as 1 pmol. (b) DTB synthesis specifically required malonyl-CoA as substrate. Structurally related compounds such as acetyl-CoA, succinyl-CoA and malonate were inactive. (c) DTB synthesis required malonyl-CoA, BioC, NADPH and all other reactions components as given for in vitro DTB synthesis in Methods. When pimeloyl-ACP methyl ester was added, both BioC and malonyl-CoA were omitted.
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Figure 2: Requirements for DTB synthesis from malonyl-CoAThe cell free extract was prepared from the ΔbioC strain STL96 which carries multiple copies of all the bio operon genes except bioC. Purified and refolded BioC was added to all reactions except as shown. Bioassays were done using strain ER90 (ΔbioF) as described in Methods. (a) Standard DTB was bioassayed and reliably detected in levels as low as 1 pmol. (b) DTB synthesis specifically required malonyl-CoA as substrate. Structurally related compounds such as acetyl-CoA, succinyl-CoA and malonate were inactive. (c) DTB synthesis required malonyl-CoA, BioC, NADPH and all other reactions components as given for in vitro DTB synthesis in Methods. When pimeloyl-ACP methyl ester was added, both BioC and malonyl-CoA were omitted.

Mentions: DTB synthesis was determined by bioassay with E. coli strain ER90 (ΔbioF bioC bioD) which carries an insertion-deletion mutation within bioF that also inactivates the downstream genes, bioC and bioD, by transcriptional polarity5,20. Hence, strain ER90 is defective in synthesis of KAPA, DAPA and DTB, but proficient in conversion of DTB to biotin20. Detection by bioassay was required because biotin synthesis is an extremely low capacity biosynthetic pathway (E. coli requires only about 100 biotin molecules per cell). In the bioassay, which can reliably detect 1 ρmol of DTB (Fig. 2a), the test solution diffuses from a filter disk into biotin-free minimal medium agar seeded with an appropriate DTB (or biotin)-requiring E. coli strain. If grow proceeds a redox indicator becomes reduced and forms a bright red, insoluble deposit with the area of the red spot being proportional to the concentration of the biotin pathway intermediate21.


Biotin synthesis begins by hijacking the fatty acid synthetic pathway.

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

Requirements for DTB synthesis from malonyl-CoAThe cell free extract was prepared from the ΔbioC strain STL96 which carries multiple copies of all the bio operon genes except bioC. Purified and refolded BioC was added to all reactions except as shown. Bioassays were done using strain ER90 (ΔbioF) as described in Methods. (a) Standard DTB was bioassayed and reliably detected in levels as low as 1 pmol. (b) DTB synthesis specifically required malonyl-CoA as substrate. Structurally related compounds such as acetyl-CoA, succinyl-CoA and malonate were inactive. (c) DTB synthesis required malonyl-CoA, BioC, NADPH and all other reactions components as given for in vitro DTB synthesis in Methods. When pimeloyl-ACP methyl ester was added, both BioC and malonyl-CoA were omitted.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2925990&req=5

Figure 2: Requirements for DTB synthesis from malonyl-CoAThe cell free extract was prepared from the ΔbioC strain STL96 which carries multiple copies of all the bio operon genes except bioC. Purified and refolded BioC was added to all reactions except as shown. Bioassays were done using strain ER90 (ΔbioF) as described in Methods. (a) Standard DTB was bioassayed and reliably detected in levels as low as 1 pmol. (b) DTB synthesis specifically required malonyl-CoA as substrate. Structurally related compounds such as acetyl-CoA, succinyl-CoA and malonate were inactive. (c) DTB synthesis required malonyl-CoA, BioC, NADPH and all other reactions components as given for in vitro DTB synthesis in Methods. When pimeloyl-ACP methyl ester was added, both BioC and malonyl-CoA were omitted.
Mentions: DTB synthesis was determined by bioassay with E. coli strain ER90 (ΔbioF bioC bioD) which carries an insertion-deletion mutation within bioF that also inactivates the downstream genes, bioC and bioD, by transcriptional polarity5,20. Hence, strain ER90 is defective in synthesis of KAPA, DAPA and DTB, but proficient in conversion of DTB to biotin20. Detection by bioassay was required because biotin synthesis is an extremely low capacity biosynthetic pathway (E. coli requires only about 100 biotin molecules per cell). In the bioassay, which can reliably detect 1 ρmol of DTB (Fig. 2a), the test solution diffuses from a filter disk into biotin-free minimal medium agar seeded with an appropriate DTB (or biotin)-requiring E. coli strain. If grow proceeds a redox indicator becomes reduced and forms a bright red, insoluble deposit with the area of the red spot being proportional to the concentration of the biotin pathway intermediate21.

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