Val, D.Chapman-Smith, A.Walker, M.Cronan, J.Wallace, J.2006-07-052006-07-051995Biochemical Journal, 1995; 312(3):817-8250264-60211470-8728http://hdl.handle.net/2440/11269In Saccharomyces cerevisiae there are two isoenzymes of pyruvate carboxylase (Pyc) encoded by separate genes designated PYC1 and PYC2. We report the isolation and sequencing of a PYC2 gene, and the localization of both genes on the physical map of S. cerevisiae. Comparison with the previously reported sequence [Stucka, Dequin, Salmon and Gancedo (1991) Mol. Gen. Genet. 229, 307-315] revealed significant differences within the open reading frame. The most notable difference was near the 3' end, where we found a single base deletion reducing the open reading frame by 15 bases. We have confirmed the C-terminus of Pyc2 encoded by the gene isolated here by expressing and purifying an 86-amino-acid biotin-domain peptide. In addition, we investigated the effects of the two changes in the Pyc2 biotin domain (K1155R substitution and Q1178P/five-amino-acid extension) on the extent of biotinylation in vivo by Escherichia coli biotin ligase, and compared the biotinylation of peptides containing these changes with that of two different-length Pyc1 biotin-domain peptides. The K1155R substitution had very little effect on biotinylation, but the five-amino-acid C-terminal extension to Pyc2 and the N-terminal extension to Pycl both improved biotinylation in vivo.enEscherichia coliSaccharomyces cerevisiaeBiotinIsoenzymesPyruvate CarboxylaseDNA, FungalChromosome MappingSequence AnalysisMutagenesisBinding SitesAmino Acid SequenceBase SequenceSequence HomologyPolymorphism, GeneticGenes, FungalMolecular Sequence DataJournal article0030004420001995157710.1042/bj3120817A1995TM609000262-s2.0-002959341368426Walker, M. [0000-0002-6934-3787]