title: The moonlighting function of pyruvate carboxylase resides in the non-catalytic end of the TIM barrel.
abstract: Pyruvate carboxylase is a highly conserved enzyme that functions in replenishing the tricarboxylic acid cycle with oxaloacetate. In the yeast Hansenulapolymorpha, the pyruvate carboxylase protein is also required for import and assembly of the peroxisomal enzyme alcohol oxidase. This additional role, which is unrelated to the enzyme activity, represents an example of a special form of multifunctionality called moonlighting. We have performed a detailed site-directed mutagenesis approach to elucidate which region(s) of H. polymorpha pyruvate carboxylase are involved in its second function. This resulted in the identification of three amino acids that are essential for the moonlighting function. Mutating these residues in a single mutant protein fully inactivated the moonlighting function, but not the enzyme activity of pyruvate carboxylase because the strain was prototrophic. A 3D homology model revealed that all three residues are positioned at the side of a TIM barrel where the N-terminal ends of the beta-strands are located. This is a novel observation as the TIM barrel proteins invariably are enzymes and have their catalytic side at the C-terminal end of the beta-sheets. Our finding implies that a TIM barrel fold can also fulfill a non-enzymatic function and that this function can reside at the N-terminal end of the barrel.
authors.affiliation: Department of Molecular Cell Biology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), Kluyver Centre for Genomics of Industrial Fermentation, University of Groningen, NL-9750 AA Haren, The Netherlands.
authors.forename: Daphne H E W
authors.initials: DH
authors.lastname: Huberts
authors.forename: Hanka
authors.initials: H
authors.lastname: Venselaar
authors.forename: Gert
authors.initials: G
authors.lastname: Vriend
authors.forename: Marten
authors.initials: M
authors.lastname: Veenhuis
authors.forename: Ida J
authors.initials: IJ
authors.lastname: van der Klei
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D004789:Enzyme Activation / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D030781:Organisms, Genetically Modified; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D011766:Pyruvate Carboxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D013329:Structure-Activity Relationship
chemical_list: D005656:Fungal Proteins; D011766:Pyruvate Carboxylase