Transcriptional regulation and enzymatic functions of the Escherichia coli biotin protein ligase
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 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 electromobility shift assays. Surprisingly, although most of the mutations were located in the catalytic domain, all those tested excepting 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 shows that normal ligase activity does not ensure normal DNA binding. The BirA biotin protein ligase of E. coli belongs to the winged helix-turn-helix (wHTH) family of transcriptional regulators. The N-terminal BirA domain is required for both transcriptional regulation of biotin synthesis and biotin protein ligase activity. In Chapter 3, I addressed the structural and functional role of the wing of the wHTH motif in both BirA functions. A panel of N-terminal deletion mutant proteins, including a discrete deletion of the wing motif, were unable to bind DNA. However, all the N- terminal deletion mutants weakly complemented growth of a ΔbirA strain at low biotin concentrations indicating compromised ligase activity. A wing domain chimera was constructed by replacing the BirA wing with the nearly isosteric wing of the E. coli OmpR transcription factor. Although this chimera BirA was defective in operator binding, it was much more efficient in complementation of a ΔbirA strain than was the wing-less protein. The enzymatic activities of the wing deletion and chimera proteins in the in vitro synthesis of biotinoyl-5ʹ-AMP differed greatly. The wing deletion BirA accumulated an off-pathway compound, ADP, whereas the chimera protein did not. Moreover, a single residue alteration in the wing bypasses the deleterious effects caused by mutations in the biotin-binding loop of the ligase active site. I believe that the role of the wing in the BirA enzymatic reaction is to orient the active site and thereby protect biotinoyl-5ʹ-AMP from attack by solvent. This is the first evidence that the wing domain of a wHTH protein can play an important role in enzymatic activity.
Biotin Protein Ligase, Biotin, Transcription repressor/regulator
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign