title: A distinct holoenzyme organization for two-subunit pyruvate carboxylase.
abstract: Pyruvate carboxylase (PC) has important roles in metabolism and is crucial for virulence for some pathogenic bacteria. PC contains biotin carboxylase (BC), carboxyltransferase (CT) and biotin carboxyl carrier protein (BCCP) components. It is a single-chain enzyme in eukaryotes and most bacteria, and functions as a 500 kD homo-tetramer. In contrast, PC is a two-subunit enzyme in a collection of Gram-negative bacteria, with the α subunit containing the BC and the β subunit the CT and BCCP domains, and it is believed that the holoenzyme has α4β4stoichiometry. We report here the crystal structures of a two-subunit PC from Methylobacillus flagellatus. Surprisingly, our structures reveal an α2β4stoichiometry, and the overall architecture of the holoenzyme is strikingly different from that of the homo-tetrameric PCs. Biochemical and mutagenesis studies confirm the stoichiometry and other structural observations. Our functional studies in Pseudomonas aeruginosa show that its two-subunit PC is important for colony morphogenesis.
authors.affiliation: Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
authors.forename: Philip H
authors.initials: PH
authors.lastname: Choi
authors.affiliation: Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
authors.forename: Jeanyoung
authors.initials: J
authors.lastname: Jo
authors.affiliation: Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
authors.forename: Yu-Cheng
authors.initials: YC
authors.lastname: Lin
authors.affiliation: Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 112, Taiwan.
authors.forename: Min-Han
authors.initials: MH
authors.lastname: Lin
authors.affiliation: Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 112, Taiwan.
authors.forename: Chi-Yuan
authors.initials: CY
authors.lastname: Chou
authors.affiliation: Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
authors.forename: Lars E P
authors.initials: LEP
authors.lastname: Dietrich
authors.affiliation: Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
authors.forename: Liang
authors.initials: L
authors.lastname: Tong
mesh_terms: D000103:Acetyl-CoA Carboxylase / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry*; D001710:Biotin / Q000737:chemistry; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism; D054889:Fatty Acid Synthase, Type II / Q000737:chemistry; D017353:Gene Deletion; D020035:Holoenzymes; D020631:Methylobacillus / Q000201:enzymology*; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D011487:Protein Conformation; D000072417:Protein Domains; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D011766:Pyruvate Carboxylase / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D020035:Holoenzymes; D001710:Biotin; D054889:Fatty Acid Synthase, Type II; D019731:Carbon-Nitrogen Ligases; C019791:biotin carboxylase; D011766:Pyruvate Carboxylase; D000103:Acetyl-CoA Carboxylase; C079633:biotin carboxyl carrier protein