Deletion variants of L-hydroxyisocaproate dehydrogenase--probing substrate specificity

The substrate specificity and catalytic activity of the dinucleotide-dependent L-2-hydroxyisocaproate dehydrogenase from Lactobacillus confusus (L-HicDH) have been altered by modifying an enzyme region which is assumed to be involved in substrate recognition. The design of the variant enzymes was based on an amino acid alignment of the modified region with the functionally related L-lactate dehydrogenases. The best absolute sequence similarity for a protein with known tertiary structure was found for L-lactate dehydrogenase from dogfish (23%). In this study, the coenzyme loop, a functional element which is essential for catalysis and substrate specificity, was modified in order to identify the residues involved in the catalytic reaction and observe the effect on the substrate specificity. Deletions were introduced into the L-hydroxyisocaproate gene by site-directed mutagenesis. Several deletion-variant enzymes Ile100A delta, Lys100B delta, Leu101 delta, Asn105A delta and Pro105 B delta showed an altered substrate specificity. For the variant enzyme with the deletion of Asn/Pro105A/ B, 2-oxo carboxylic acids branched at C4 proved to be better substrates than 2-oxocaproate, the substrate with the best k(cat)/Km ratio known for the wild-type enzyme. The mutation resulted in a 5.2-fold increased catalytic efficiency towards 2-oxoisocaproate compared to the wild-type enzyme. After deleting Ile/Lys100A/B, 2-phenylpyruvate is the only substrate which is still converted at a significant catalytic rate. The kcat ratios of 2-oxocaproate versus 2-phenylpyruvate changed by a factor of 6500 when comparing wild-type enzyme and deletion-variant enzyme data. The single amino acid deletions in position 100A and 100B caused drastic reductions in the catalytic activity for all tested substrates, whereas the deletion of Lys100B, Leu101, Asn105A as well as Pro105B showed more specific modifications in catalytic rates and substrate recognition for each tested substrate.