The synthesis, regulation and function of biotin in bacteria

Song, Xuejiao

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https://hdl.handle.net/2142/115359 Copy

Description

Title

The synthesis, regulation and function of biotin in bacteria

Author(s)

Song, Xuejiao

Issue Date

2022-04-06

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

Cronan, John E

Doctoral Committee Chair(s)

Cronan, John E

Committee Member(s)

• Imlay, James A.
• Sligar, Stephen G
• Jin, Hong

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• biotin synthesis
• biotin protein ligase
• acetyl-CoA carboxylase
• transcriptional regulation

Abstract

Biotin, also known as vitamin H, is essential for all three domains of life. Biotin acts as an essential cofactor for biotin-dependent enzymes, including carboxylases, decarboxylases and transcarboxylases. These enzymes play essential roles in central metabolic pathways, such as fatty acid synthesis, amino acid metabolism and polyketide biosynthesis. De novo biotin biosynthesis is widespread in microorganisms, fungi and plants, but is absent in mammals and birds. Biotin synthesis is a metabolically expensive process, requiring as many as 20 equivalents of ATP and at least 5 enzymes to assemble one molecule of biotin. Bacterial biotin synthetic genes are generally found clustered into bio-operons to facilitate tight regulation. Dethiobiotin synthetase (DTBS) catalyzes the penultimate step of biotin biosynthesis, the formation of 7,8-diaminononanoate (DAPA). In Escherichia coli, DTBS is encoded by the bio-operon gene bioD. Several studies have reported transcriptional activation of ynfK, a gene of unknown function, under anaerobic conditions. Alignment of YnfK with BioD leads to the suggestion that YnfK has DTBS activity. I report that YnfK is a functional DTBS, although an enzyme of poor activity that is poorly expressed. Supplementation of growth medium with DAPA or substitution of BioD active site residues for the corresponding YnfK residues greatly improved the DTBS activity of YnfK. I confirmed that FNR activates transcriptional level of ynfK during anaerobic growth and identified the FNR binding site of ynfK. The ynfK gene is well conserved in γ-proteobacteria. Biotinylation refers to the extremely specific process whereby biotin becomes covalently attached to a single lysine residue of its cognate biotin receptor protein(s), the process of which is catalyzed by biotin protein ligase (BPL). Protein biotinylation requires tight regulation since the biotinylation process is involved in protein signaling, localization, activation and degradation. Three classes of BPLs have been reported thus far, and class I and class II BPLs are generally found in bacteria. Class I BPLs contain the central catalytic domain and the C-terminal cap and only catalyze the biotinylation process. In contrast Group II BPLs have an additional N-terminal DNA binding domain that functions not only in biotinylation but also in transcriptional regulation of genes of biotin biosynthesis and transport. Most bacteria contain only one biotin protein ligase whereas Clostridium acetobutylicum contains two biotin protein ligase homologs: BplA and BirA’. Sequence alignments show that BplA is a typical group I BPL whereas BirA’ lacks the C terminal domain conserved throughout extant BPL proteins. This raised the questions of why two BPL homologs are needed and why the apparently defective BirA’ has been retained. I have used in vivo and in vitro assays to show that BplA is a functional BPL whereas BirA’ acts as a biotin sensor involved in transcriptional regulation of biotin transport. I also successfully converted BirA’ into a functional biotin protein ligase with regulatory activity by fusing it to the C-terminal domain from BplA. Finally, I provided evidence that BplA and BirA’ interact in vivo. Acetyl-CoA carboxylase (ACC) is a biotin-dependent enzyme and catalyzes the first reaction in fatty acid biosynthesis, the formation of malonyl-CoA from acetyl-CoA. In Escherichia coli, ACC is a very unstable complex made of four subunits with a defined stoichiometry of: 2AccA-4AccB-2AccC-2AccD. The genes accB and accC are co-transcribed, whereas the accA and accD genes are distant from the accBC operon and from one another. This raises the question of how the defined stoichiometry is achieved. Early studies have shown that expression of the ACC subunits is growth dependent, which is related to the stringent response initiated by ppGpp and DksA. Later studies show that the FadR transcription factor binds the accA and accB promoters and activates transcription. In this study, I used in vitro transcription assays to show that ppGpp and DksA directly inhibit transcription of the acc genes. The effect of FadR on acc transcription was re-examined using both EMSA and in vitro transcription assays. Moreover, I found that the nucleoid-associated protein, H-NS, is important for accB expression. Surprisingly, FabR, a transcriptional factor generally considered to only regulate the genes of unsaturated fatty acid synthesis, inhibited acc gene transcription.

Graduation Semester

2022-05

Type of Resource

Thesis

Copyright and License Information

Copyright 2022 Xuejiao Song

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