, respectively, at the final step of anaerobic glycolysis, concomitantly oxidizing NADH into NAD ϩ (16). Lactic acid bacteria possess at least one of the two types of LDHs, fermenting the corresponding stereoisomer of lactic acid (12). In spite of the similarity in their catalytic reactions, the two types of enzymes are evolutionally separate from each other, belonging to distinct protein superfamilies (4,21,33). D-LDHs share a common protein structure not only with various D-2-hydroxyacid dehydrogenases (2,4,10,13,14,21,25,26,29,32,33) but also other dehydrogenases such as formate (26) and L-alanine (3) dehydrogenases.L-and D-LDHs are highly divergent enzymes in lactic acid bacteria, showing great variety in both their amino acid sequences and catalytic properties or substrate specificities (1,2,4,7,12,21,33). There is only 40 to 50% amino acid identity among known D-LDHs of different Lactobacillus species, which show significantly different kinetic parameters, such as k cat and K m for substrates (4,7,21,33). Instead of or together with D-LDH, some lactobacilli such as Lactobacillus casei (17,19,25) and L. delbrueckii (5) have D-hydroxyisocaproate dehydrogenases (D-HicDHs), which exhibit high activity not toward pyruvate but 2-ketoacids with larger aliphatic or aromatic side chains at the C-3 position, while L. confusus has L-HicDH, an L-LDH-related enzyme (30). D-HicDHs show 40 to 50% amino acid identity with known Lactobacillus D-LDHs (5, 25, 33), which is comparable to the identity among the D-LDHs, suggesting that these two types of enzymes are particularly related evolutionally. Since optically active 2-hydroxyacids are valuable for the synthesis of useful compounds (17-19), D-HicDHs are promising enzymes for industrial application, although their actual physiological role remains uncertain.The substrate recognition by an enzyme has been extensively studied in the case of L-LDH, mostly through alteration of the substrate specificity by means of protein engineering (1,8,11,15,37,38), but much less has been learnt about D-LDH or related enzymes. Recently, however, the three-dimensional structures of L. pentosus apo (32) and L. bulgaricus holo (27) D-LDHs, and the ternary complex of L. casei D-HicDH (10) were determined, implying their substrate recognition sites. Figure 1 shows the position of Leu51 in the substrate binding site of the L. casei D-HicDH ternary complex structure (10), together with those of Arg234, Glu263, and His294, which were indicated to be residues involved in the catalytic function of D-LDH by amino acid replacement studies (20,22,(33)(34)(35). Since Leu51 is located very near the substrate C-3 position and is consistently replaced by conserved Tyr (Tyr52) in Lactobacillus 21,33), it is easily imaginable that the amino acid at this position defines the size or shape of the hydrophobic pocket for the C-3 groups of 2-ketoacid substrates (10). In this paper, we show how L. pentosus D-LDH is sufficiently converted into a D-HicDH through only 1 amino acid replacement of Tyr52 to Leu.An olig...