| dc.description.abstract | Biotin (vitamin H), an essential coenzyme synthesized by plants and most prokaryotes, is required by all organisms. Biotin serves as a cofactor for enzymes that participates in carbon dioxide transfer. Biotin protein ligase (BPL), also known as holocarboxylase synthetase (HCS) or BirA, is the enzyme responsible for attaching biotin to proteins. Although this modification is ubiquitous and necessary for all organisms, a relatively rare post-translational modification. Only one to five protein acceptors in every single species. BPL catalyzes the attachment of biotin to a specific lysine residue located within a conserved sequence AMKM. Arc1p is a non-specific tRNA-binding protein of Saccharomyces cerevisiae. It forms a ternary complex in the cytoplasm with glutamyl-tRNA synthetase and methionyl-tRNA synthetase, and enhances the aminoacylation activity of these two enzymes. In addition, Arc1p also regulates the intracellular distributions of these two enzymes. While Arc1p lacks an AMKM motif, it can be specifically biotinylated by BPL1. In contrast, BPL1s of most yeast species cannot biotinylate their own Arc1p homologues. Recent studies have found that Arc1p contains a unique biotinylation site, SSKD. The aim of this project is to map the substrate-specific site of yeast BPL. According to the crystal structure models in Protein Data Bank, the structures of AMKM and SSKD are quite different. Site-directed Mutagenesis further showed that amino acid sequences flanking SSKD are important for biotinylation, and altering these amino acids significantly reduces the biotinylation level. Arc1p could not be biotinylated when its SSKD was mutated to AMKM. Also, we found that there are some unique embedded peptides in the C-terminal domain of S. cerevisiae BPL1. These unique peptides are absent ftrom most yeast BPL1 homologues. However, deleting these unique peptide sequences did not affect its ability to biotinylate Arc1p. In addition, exchange of N and C domains between BPL1s of different yeast species failed to locate the substrate-specific region accurately. Further research is underway to map the substrate-specific site of yeast BPL1. | en_US |