A Specific, Highly Active Malate Dehydrogenase by Redesign of a Lactate Dehydrogenase Framework

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: Asp 197 → Asn, Thr 246 → Gly, and Gln 102 → Arg). Each modification shifts the specificity from lactate to malate, although only the last (Gln 102 → Arg) provides an effective and highly specific catalyst for the new substrate. This synthetic enzyme has a ratio of catalytic rate ( k cat ) to Michaelis constant ( K m ) 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 .