Altered Regulation of Escherichia coli Biotin Biosynthesis in BirA Superrepressor Mutant Strains

Abstract

Transcription of the Escherichia 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. Although several aspects of this regulatory system are well understood, no BirA superrepressor mutant strains had been isolated. 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. We isolated mutant strains having this phenotype by a combined selection-screening approach and resolved multiple mutations to give several birA superrepressor alleles, each having a single mutation, all of which showed repression dominant over that of 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 strains except that encoding G154D BirA showed derepression of bio operon transcription upon overproduction of a biotin-accepting protein. In BirA, G154D was a lethal mutation in single copy, and the purified protein was unable to transfer biotin from enzyme-bound biotinoyl-adenylate either to the natural acceptor protein or to a biotin-accepting peptide sequence. Consistent with the transcriptional repression data, each of the purified mutant proteins showed increased affinity for the biotin operator DNA in electrophoretic mobility shift assays. Surprisingly, although most of the mutations were located in the catalytic domain, all of those tested, except G154D BirA, had normal ligase activity. Most of the mutations that gave superrepressor phenotypes altered residues located close to the dimerization interface of BirA. However, two mutations were located at sites well removed from the interface. The properties of the superrepressor mutants strengthen and extend other data indicating that BirA function entails extensive interactions among the three domains of the protein and show that normal ligase activity does not ensure normal DNA binding. In Escherichia coli, the synthesis of the essential enzyme cofactor biotin is regulated at the transcriptional level by an unusual repressor called BirA. BirA is unusual in that it is also an essential metabolic enzyme, the sole biotin protein ligase of this bacterium. Biotin protein ligases are found throughout the three domains of life and catalyze the covalent attachment of biotin to its cognate acceptor proteins that play key roles in central metabolism. The fact that BirA (a protein of 35.3 kDa) is both an enzyme and a transcriptional repressor makes biotin (bio) operon transcription sensitive not only to the intracellular concentration of biotin, but also to the levels of cognate...

Brucella BioR Regulator Defines a Complex Regulatory Mechanism for Bacterial Biotin Metabolism

“…Most of our current knowledge on regulation of bacterial biotin metabolism comes from studies with E. coli (1,17). Biotin protein ligase (BPL) is referred to as a universal enzyme widespread throughout all forms of life which covalently attaches biotin to its cognate acceptor proteins, such as the AccB subunit (also called the biotin carboxyl carrier protein [BCCP]) of acetyl-CoA carboxylase (ACC), which catalyzes the first committed step of fatty acid biosynthesis (17).…”

“…Biotin protein ligase (BPL) is referred to as a universal enzyme widespread throughout all forms of life which covalently attaches biotin to its cognate acceptor proteins, such as the AccB subunit (also called the biotin carboxyl carrier protein [BCCP]) of acetyl-CoA carboxylase (ACC), which catalyzes the first committed step of fatty acid biosynthesis (17). The E. coli birA gene product is a model BPL that has undergone extensive investigations, ranging from genetics (18)(19)(20) and biochemistry (21)(22)(23)(24)(25) to structural biology/biophysics (21,26,27).…”

Genetic, Genomic, and Transcriptomic Studies of Pyruvate Metabolism in Methanosarcina barkeri Fusaro

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“…Transcription of the biotin operon is directly regulated by a biotin-protein ligase, BirA, which is an enzyme protein that catalyzes the covalent attachment of biotin to its acceptor proteins (12). The operon transcription is sensitive not only to the intracellular biotin concentration, but also to the levels of cognate proteins that require biotin as a cofactor (13).…”