Biotin synthesis in α-proteobacteria

Hu, Yuanyuan

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https://hdl.handle.net/2142/114074 Copy

Description

Title

Biotin synthesis in α-proteobacteria

Author(s)

Hu, Yuanyuan

Issue Date

2021-12-03

Director of Research (if dissertation) or Advisor (if thesis)

Cronan, John E.

Doctoral Committee Chair(s)

Cronan, John E.

Committee Member(s)

• Gennis, Robert B
• Imlay, James A
• Hong, Jin

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Date of Ingest

2022-04-29T21:58:25Z

Keyword(s)

• Biotin
• pimelic acid
• pimeloyl-ACP
• bioZ
• glutaric acid
• glutaryl-CoA lysine catabolism

Abstract

Pimelic acid, a seven carbon α, ω-dicarboxylic acid (heptanedioic acid), has long been known to provide most biotin carbon atoms including all those of the valeryl side chain. Nevertheless, the first pathways of pimelate moiety synthesis have only recently been elucidated. Two mechanistically distinct pathways, those of Escherichia coli and Bacillus subtilis, are known. Both use fatty acid synthesis plus dedicated biotin synthetic enzymes to assemble the pimelate moiety. In contrast, the α-proteobacteria, which include important plant and mammalian pathogens plus plant symbionts, lack the E. coli and B. subtilis pimelate synthesis genes and instead encode a putative 3-ketoacyl-ACP synthase enzyme called BioZ in their biotin gene clusters. We report a novel pathway in which BioZ proteins catalyze a 3-ketoacyl-ACP synthase III-like reaction to produce pimeloyl-ACP. In this reaction five of the seven pimelate carbon atoms are derived from glutaryl-CoA, an intermediate in lysine degradation, rather than the fatty acid synthesis route used by E. coli and B. subtilis. The synthesis of glutaryl-CoA was long been thought to be a ligation of CoA to glutarate activated by ATP However such enzyme has never been identified. It was later thought to be a promiscuous conversion of oxo-adipate to glutaryl-CoA by ketoglutaryl-CoA dehydrogenase. The first publication clearly demonstrated a conversion of glutarate to glutaryl-CoA was characterized by the protein encoded by human C7orf10. The putative transferases having about 40% sequence similarity from α-proteobacteria strains and E. coli were purified and shown to catalyze transfer of CoA from succinyl-CoA to glutarate. They also have promiscuity to CoA donor and receptor with different chain length. Finally, disruption of the caiB gene resulted in biotin auxotrophy in Agrobacterium tumefaciens C58 and limited biotin synthesis in E. coli ΔbioC ΔbioH double deletion strains supplemented with bioZ. Structure and substrate specificity of BioZ has also been studied during this thesis work. The BioZ proteins, like the 3-ketoacyl-ACP synthase III fatty acid synthesis proteins (FabHs) use a Cys-His-Asn triad to catalyze the Claisen-like condensation reaction. Mutation of the catalytic cysteine assigned by sequence alignment to alanine resulted in an inactive enzyme and biotin auxotrophy in A. tumefaciens. The positively charged guanidino group of an arginine 147 residues in BioZ is pointing towards the negatively charged carboxyl group of glutaryl-CoA in the crystal structure of this mutant protein, and thus seems likely to function to stabilize the negative charge within the tunnel. However, mutation of the analogous threonine to arginine in E. coli FabH didn’t complement the pimeloyl-ACP auxotroph E. coli MG1655 ΔbioC ΔbioH double deletion strain for growth. Arginine 147 is likely not sufficient to determine substrate specificity in BioZ.

Graduation Semester

2021-12

Type of Resource

Thesis

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http://hdl.handle.net/2142/114074

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Copyright 2021 Yuanyuan Hu

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