Transcriptional regulation and enzymatic functions of the Escherichia coli biotin protein ligase
type: CreativeWork
description: Biotin metabolism in prokaryotes is directly linked to transcriptional regulation by the enzyme biotin protein ligase (BPL). This system is most thoroughly studied and characterized in Escherichia coli. Transcription of the E. coli biotin (bio) operon is directly regulated by the biotin-protein ligase, BirA, the enzyme that covalently attaches biotin to its cognate acceptor proteins. Binding of BirA to the bio operator requires dimerization of the protein, which is triggered by BirA-catalyzed synthesis of biotinoyl- adenylate (biotinoyl-5'-AMP), the obligatory intermediate of the ligation reaction. In this thesis I carried out an in-depth analysis of the BirA protein from E. coli using a combination of mutational and biochemical analysis. These studies provide the first strong evidence that the inter-domain interactions are required for full enzymatic and transcriptional functions of this highly dynamic enzyme. In Chapter 2, I describe the isolation and characterization of a new class of superrepressor mutant strains of BirA. Such superrepressor BirA proteins would repress the biotin operon transcription in vivo at biotin concentrations well below those needed for repression by wild-type BirA. Mutant strains having this phenotype were isolated by a combined selection-screening approach. The multiple mutations were resolved to give several birA superrepressor alleles each having a single mutation all of which showed repression dominant over the wild-type allele. All of these mutant strains repressed bio operon transcription in vivo at biotin concentrations that gave derepression of the wild- type strain and retained sufficient ligation activity for growth when overexpressed. All of the superrepressor strains except that encoding G154D BirA showed derepression of bio operon transcription upon overproduction of a biotin accepting protein. The G154D BirA was a lethal mutation in single copy and the purified protein was unable to transfer biotin from enzyme bound biotino…
abstract: Biotin metabolism in prokaryotes is directly linked to transcriptional regulation by the enzyme biotin protein ligase (BPL). This system is most thoroughly studied and characterized in Escherichia coli. Transcription of the E. coli biotin (bio) operon is directly regulated by the biotin-protein ligase, BirA, the enzyme that covalently attaches biotin to its cognate acceptor proteins. Binding of BirA to the bio operator requires dimerization of the protein, which is triggered by BirA-catalyzed synthesis of biotinoyl- adenylate (biotinoyl-5'-AMP), the obligatory intermediate of the ligation reaction. In this thesis I carried out an in-depth analysis of the BirA protein from E. coli using a combination of mutational and biochemical analysis. These studies provide the first strong evidence that the inter-domain interactions are required for full enzymatic and transcriptional functions of this highly dynamic enzyme. In Chapter 2, I describe the isolation and characterization of a new class of superrepressor mutant strains of BirA. Such superrepressor BirA proteins would repress the biotin operon transcription in vivo at biotin concentrations well below those needed for repression by wild-type BirA. Mutant strains having this phenotype were isolated by a combined selection-screening approach. The multiple mutations were resolved to give several birA superrepressor alleles each having a single mutation all of which showed repression dominant over the wild-type allele. All of these mutant strains repressed bio operon transcription in vivo at biotin concentrations that gave derepression of the wild- type strain and retained sufficient ligation activity for growth when overexpressed. All of the superrepressor strains except that encoding G154D BirA showed derepression of bio operon transcription upon overproduction of a biotin accepting protein. The G154D BirA was a lethal mutation in single copy and the purified protein was unable to transfer biotin from enzyme bound biotino…
author: Chakravartty, Vandana
language: en
keywords: Biotin Protein Ligase, Biotin, Transcription repressor/regulator
date published: 2015-01-21T19:55:28Z
date modified: 2026-07-22T22:26:07Z
provider: University of Illinois - Urbana-Champaign
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Illinois at Urbana-Champaign

PropertyValue
property ID: Handle
value: http://hdl.handle.net/2142/72997