Designs for a broad substrate specificity keto acid dehydrogenase

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Abstract
Variations have been made to the structure of the nicotinamide adenine dinucleotide (NAD) dependent L-lactate dehydrogenase from Bacillus stearothermophilus at regions of the enzyme that we believe determine specificity toward different .alpha.-hydroxy acids (RCHOHCOO-, R = CH3, C2H5, etc.). Two regions of LDH that border the active site (but are not involved in the catalytic reaction) were altered in order to accomodate substrates with hydrophobic side chains larger than that of the naturally preferred substrate, pyruvate (R = CH3). The mutations 102-105GlnLysPro .fwdarw. MetValSer and 236-237AlaAla .fwdarw. GlyGly were made to increase the tolerance for large hydrophobic substrate side chains. The triple and double mutants alone gave little improvement for branched-chain-substituted pyruvates. The five changes together produced a broader substrate specificity .alpha.-hydroxy acid dehydrogenase, with a 55-fold improved kcat for .alpha.-ketoisocaproate to a value about 1/14 that of the native enzyme for pyruvate. Rational protein engineering enabled coupled changes in enzyme structure to be obtained with greater probability of success than random mutagenesis.