title: Rational construction of a 2-hydroxyacid dehydrogenase with new substrate specificity.
abstract: Using site-directed mutagenesis on the lactate dehydrogenase gene from Bacillus stearothermophilus, three amino acid substitutions have been made at sites in the enzyme which we suggest in part determine specificity toward different hydroxyacids (R-CHOH-COOH). To change the preferred substrates from the pyruvate/lactate pair (R = -CH3) to the oxaloacetate/malate pair (R = -CH2-COO-), the volume of the active site was increased (thr 246----gly), an acid was neutralized (asp-197----asn) and a base was introduced (gln-102 - greater than arg). The wild type enzyme has a catalytic specificity for pyruvate over oxaloacetate of 1000 whereas the triple mutant has a specificity for oxaloacetate over pyruvate of 500. Despite the severity and extent of these active site alterations, the malate dehydrogenase so produced retains a reasonably fast catalytic rate constant (20 s-1 for oxaloacetate reduction) and is still allosterically controlled by fructose-1,6-bisphosphate.
authors.affiliation: Department of Biochemistry, University of Bristol Medical School, U.K.
authors.forename: A R
authors.initials: AR
authors.lastname: Clarke
authors.forename: C J
authors.initials: CJ
authors.lastname: Smith
authors.forename: K W
authors.initials: KW
authors.lastname: Hart
authors.forename: H M
authors.initials: HM
authors.lastname: Wilks
authors.forename: W N
authors.initials: WN
authors.lastname: Chia
authors.forename: T V
authors.initials: TV
authors.lastname: Lee
authors.forename: J J
authors.initials: JJ
authors.lastname: Birktoft
authors.forename: L J
authors.initials: LJ
authors.lastname: Banaszak
authors.forename: D A
authors.initials: DA
authors.lastname: Barstow
authors.forename: T
authors.initials: T
authors.lastname: Atkinson
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D045022:Lactate Dehydrogenases; D009154:Mutation; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D000429:Alcohol Oxidoreductases; D045022:Lactate Dehydrogenases; D007770:L-Lactate Dehydrogenase; C071195:D-lactate dehydrogenase; C021379:D-2-hydroxyacid dehydrogenase