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The substrate specificity and catalytic activity of the dinucleotide-dependent L-2-hydroxyisocaproate dehydrogenase from Lactobacillus confisus (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 IlelOOAd, LyslOOBd, LeulOld, Asnl05Ad and ProlOSBd showed an altered substrate specificity. For the variant enzyme with the deletion of Asn/ProlOSA/ B, 2-0x0 carboxylic acids branched at C4 proved to be better substrates than 2-oxocaproate, the substrate with the best kc,/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/LyslOOA/B, 2-phenylpyruvate is the only substrate which is still converted at a significant catalytic rate. The k,,, 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 lOOA and 100B caused drastic reductions in the catalytic activity for all tested substrates, whereas the deletion of LyslOOB, LeulO1, AsnlOSA as well as ProlOSB showed more specific modifications in catalytic rates and substrate recognition for each tested substrate.L-2-hydroxyisocaproate dehydrogenase (L-HicDH) from Lactobacillus confisus was first purified and characterized by Schiitte et al. (1984). It is a NAD(H)-dependent oxidoreductase which catalyzes the reversible and stereospecific oxidation of alipathic branched and unbranched (S)-2-hydroxy carboxylic acids to 2-ox0 carboxylic acids.The active form is a homotetramer with a molecular mass of 33 kDdsubunit. In contrast to lactate dehydrogenase, which is specific for its small substrate, the preferred substrates have five or six carbon atoms. The use of this protein for the production of L-amino acids has been suggested (Wandrey et al., 1984), as a new asymmetric carbon atom is introduced during the reductive reaction. Lerch et al. (1989) reported sequence identities of more than 20% for the LHicDH sequence when compared to lactate dehydrogenases (LDH) sequences, some of which have known three-dimen-