| dc.description.abstract |
Biotin can serve as a coenzyme for a distinct set of catalytic reactions. Those biotin-dependent enzymes catalyze key steps which are involved in metabolic pathway. The covalent attachment of biotin to a conserved lysine residue in its cognate apoproteins is mediated by a specific enzyme, called biotin protein ligase (BPL). The biotinylated lysine residue is almost invariably positioned in a consensus sequence, AMKM, within the carboxylases. As a result, biotinylation can occur across widely divergent species. For example, the BPL1 homologues of yeast, Arabidopsis, and human can efficiently complement an E. coli birA mutant. Despite that, we found that some bacterial BPL1 homologues can functionally substitute for yeast BPL1, while others cannot substitute under similar conditions. To advance understanding of the functional property of this group of enzymes, we tried several different approaches, including mutagenesis, domain swapping, and gene expression. Unfortunately, none of the approaches can make the BPL rescue the negative phenotype. In addition, we tried to construct a biotin-tRNA-modifying enzyme by fusing a promiscuous Aquifex aeolicus BirA mutant and the tRNA-binding domain of yeast Arc1p. The resultant fusion protein still retained the biotin protein ligase activity, but was somehow inactive in modifying tRNA with biotin. Moreover, we have cloned BPL1 genes from ten yeast species and analyzed their sequence similarities. As it turned out, these yeast BPL1s possess 35%-60% similarities. Most interestingly, only four of the ten yeast BPL1s could biotinylate their own Arc1ps, and could biotinylate S. cerevisiae Arc1p. The remaining six yeast BPL1s failed to biotinylate their own Arc1ps, and could not biotinylate S. cerevisiae Arc1p. Such a finding may help us further delineate the substrate specificity of BPL1. | en_US |