title: Determinants of substrate specificity in KdcA, a thiamin diphosphate-dependent decarboxylase.
abstract: Thiamin diphosphate-dependent decarboxylases catalyze the non-oxidative decarboxylation of 2-keto carboxylic acids. Although they display relatively low sequence similarity, and broadly different range of substrates, these enzymes show a common homotetrameric structure. Here we describe a kinetic characterization of the substrate spectrum of a recently identified member of this class, the branched chain 2-keto acid decarboxylase (KdcA) from Lactococcus lactis. In order to understand the structural basis for KdcA substrate recognition we developed a homology model of its structure. Ser286, Phe381, Val461 and Met358 were identified as residues that appeared to shape the substrate binding pocket. Subsequently, site-directed mutagenesis was carried out on these residues with a view to converting KdcA into a pyruvate decarboxylase. The results show that the mutations all lowered the Km value for pyruvate and both the S286Y and F381W variants also had greatly increased values of k(cat) with pyruvate as a substrate.
authors.affiliation: College of Pharmacy, University of Michigan, 428 Church Street, Ann Arbor, MI 48109-1065, USA.
authors.forename: Alejandra
authors.initials: A
authors.lastname: Yep
authors.forename: George L
authors.initials: GL
authors.lastname: Kenyon
authors.forename: Michael J
authors.initials: MJ
authors.lastname: McLeish
mesh_terms: D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002262:Carboxy-Lyases; D013294:Lactococcus lactis / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D011767:Pyruvate Decarboxylase / Q000378:metabolism; D019289:Pyruvic Acid / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D019289:Pyruvic Acid; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D002262:Carboxy-Lyases; D011767:Pyruvate Decarboxylase