title: A specific, highly active malate dehydrogenase by redesign of a lactate dehydrogenase framework.
abstract: Three variations to the structure of the nicotinamide adenine dinucleotide (NAD)-dependent L-lactate dehydrogenase from Bacillus stearothermophilus were made to try to change the substrate specificity from lactate to malate: Asp197----Asn, Thr246----Gly, and Gln102----Arg). Each modification shifts the specificity from lactate to malate, although only the last (Gln102----Arg) provides an effective and highly specific catalyst for the new substrate. This synthetic enzyme has a ratio of catalytic rate (kcat) to Michaelis constant (Km) for oxaloacetate of 4.2 x 10(6)M-1 s-1, equal to that of native lactate dehydrogenase for its natural substrate, pyruvate, and a maximum velocity (250 s-1), which is double that reported for a natural malate dehydrogenase from B. stearothermophilus.
authors.affiliation: Department of Biochemistry, University of Bristol, United Kingdom.
authors.forename: H M
authors.initials: HM
authors.lastname: Wilks
authors.forename: K W
authors.initials: KW
authors.lastname: Hart
authors.forename: R
authors.initials: R
authors.lastname: Feeney
authors.forename: C R
authors.initials: CR
authors.lastname: Dunn
authors.forename: H
authors.initials: H
authors.lastname: Muirhead
authors.forename: W N
authors.initials: WN
authors.lastname: Chia
authors.forename: D A
authors.initials: DA
authors.lastname: Barstow
authors.forename: T
authors.initials: T
authors.lastname: Atkinson
authors.forename: A R
authors.initials: AR
authors.lastname: Clarke
authors.forename: J J
authors.initials: JJ
authors.lastname: Holbrook
mesh_terms: D001665:Binding Sites; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D008291:Malate Dehydrogenase / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D007770:L-Lactate Dehydrogenase; D008291:Malate Dehydrogenase