Conversion of Lactobacillus pentosus d -Lactate Dehydrogenase to a d -Hydroxyisocaproate Dehydrogenase through a Single Amino Acid Replacement

Abstract

, 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...

Distinct Conformation-mediated Functions of an Active Site Loop in the Catalytic Reactions of NAD-dependent D-Lactate Dehydrogenase and Formate Dehydrogenase

“…NADH Binding to D-LDHs-The dissociation constants (K D ) of N97D D-LDH and NADH were determined essentially according to a previous report (38). The binding of NADH to the D-LDHs was followed as the change in NADH fluorescence intensity (⌬F) essentially according to the method used for L-LDHs (39), with excitation and emission wavelengths of 340 and 460 nm, respectively, using a Jasco FP-750 spectrofluorophotometer.…”

“…⌬F was determined by comparing the fluorescence intensities of NADH in the presence and absence of the enzymes (15 M) at 30°C in 50 mM sodium MES buffer (pH 6.0). The K D values for the enzymes with NADH were calculated according to the procedure for L-LDHs (38,39) by curve fitting with Kaleidagraph. (Fig.…”

“…and NADH formed in the ternary complex. As in the case of the wild-type enzyme (38), the fluorescence intensity of NADH markedly increased when NADH was bound to the N97D enzyme (Fig. 4) (11).…”

A New Family ofD-2-Hydroxyacid Dehydrogenases That ComprisesD-Mandelate Dehydrogenases and 2-Ketopantoate Reductases

“…It has been reported that only minor structural changes, such as amino acid replacements, cause drastic change, in the enzyme function in this family, as for example, conversions from a D-lactate dehydrogenase (D-LDH) to a D-hydroxyisocaproate dehydrogenase (D-HicDH), 17,18) and even from a formate dehydrogenase to a D-2-HydDH. [19][20][21] Although the enzymes in this family generally exhibit strict specificity toward NAD, it was reported recently that Holoferax mediterranei D-2-HydDH, which belongs to the D-2-HydDH family, utilizes both NAD and NADP as coenzymes, acting on bulky 2-ketoacid substrates.…”

“…[21][22][23][24] In spite of the great variety of their catalytic functions, however, there is no reported enzyme in the D-2-HydDH family that exhibits high catalytic activity toward C3-branched substrates. 17,18,22,25) Enzymes purified from Lactobacillus curvatus, 26) Enterococcus faecalis, 27,28) and also the yeast Rhodotorula graminis 29) are known to catalyze efficiently the conversion between benzoylformate (C 6 H 5 -CO-COO À ) and D-mandelate (C 6 H 5 -CHOH-COO À ), which possess a branched side chain at the C3 position, and are called D-mandelate dehydrogenases (D-ManDHs). Nevertheless, their structural relation to the D-2-HydDH family remains uncertain, because little is known about the protein structure of D-ManDHs.…”