Potential of R-2-Hydroxyisocaproate dehydrogenase from Lactobacillus casei for stereospecific reductions

Kallwass, Helmut

doi:10.1016/0141-0229(92)90022-g

Cited by 48 publications

(17 citation statements)

References 41 publications

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“…VanH has a broader substrate specificity than the usual D-LDHs and is able to utilize 2-ketobutyrate as well as pyruvate. On the other hand, certain Lactobacilli are known to have D-2-hydroxyisocaproate dehydrogenases (D-HicDHs), which utilize still larger aliphatic or aromatic substrates than D-LDHs (7,16,18,22), although their actual physiological role is uncertain. Furthermore, the enzymes purified from Lactobacillus curvatus (17), Enterococcus faecalis (30), and the yeast Rhodotorula graminis (2,3,5,12) have been reported to efficiently catalyze the conversion between benzoylformic acid (C 6 H 5 -CO-COO Ϫ ) and D-mandelic acid (C 6 H 5 -CHOH-COO Ϫ ) and are called D-mandelate dehydrogenases (D-ManDHs) (17), although their crucial relationship to D-LDH remains uncertain (5,12).…”

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“…Furthermore, the enzymes purified from Lactobacillus curvatus (17), Enterococcus faecalis (30), and the yeast Rhodotorula graminis (2,3,5,12) have been reported to efficiently catalyze the conversion between benzoylformic acid (C 6 H 5 -CO-COO Ϫ ) and D-mandelic acid (C 6 H 5 -CHOH-COO Ϫ ) and are called D-mandelate dehydrogenases (D-ManDHs) (17), although their crucial relationship to D-LDH remains uncertain (5,12). D-LDH-related enzymes such as D-HicDHs and D-ManDHs are promising for industrial application, because optically active 2-hydroxyacids are valuable for the synthesis of useful compounds (16)(17)(18)30). In the process of screening lactic acid bacteria for D-ManDH activity, we found that E. faecalis IAM 10071 contains two distinct forms of an enzyme that exhibit marked D-ManDH activity.…”

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Two Forms of NAD-Dependent d -Mandelate Dehydrogenase in Enterococcus faecalis IAM 10071

Tamura

Ohkubo

Iwai

et al. 2002

Appl Environ Microbiol

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Two forms of NAD-dependent d-mandelate dehydrogenase (d-ManDHs) were purified from Enterococcus faecalis IAM 10071. While these two enzymes consistently exhibited high activity toward large 2-ketoacid substrates that were branched at the C3 or C4 position, they gave distinctly different Km and V max values for these substrates and had distinct molecular weights by gel electrophoresis and gel filtration.

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Two Forms of NAD-Dependent d -Mandelate Dehydrogenase in Enterococcus faecalis IAM 10071

Tamura

Ohkubo

Iwai

et al. 2002

Appl Environ Microbiol

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“…These NAD(H)-dependent oxidoreductases are of interest in a variety of fields. Firstly, they are valuable catalysts for the production of the stereospecific isomers of 2-hydroxyacids that are used in the production of semisynthetic antibiotics or pharmaceuticals (32). Secondly, in lactic acid bacteria, hydroxyacid dehydrogenases are believed to be negatively involved in flavor production, since they compete with other enzymes generating flavor compounds from 2-keto acids derived from amino acids (66), while 2-hydroxyacids are not aroma compounds or precursors of flavor compounds.…”

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The d -2-Hydroxyacid Dehydrogenase Incorrectly Annotated PanE Is the Sole Reduction System for Branched-Chain 2-Keto Acids in Lactococcus lactis

et al. 2009

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Hydroxyacid dehydrogenases of lactic acid bacteria, which catalyze the stereospecific reduction of branchedchain 2-keto acids to 2-hydroxyacids, are of interest in a variety of fields, including cheese flavor formation via amino acid catabolism. In this study, we used both targeted and random mutagenesis to identify the genes responsible for the reduction of 2-keto acids derived from amino acids in Lactococcus lactis. The gene panE, whose inactivation suppressed hydroxyisocaproate dehydrogenase activity, was cloned and overexpressed in Escherichia coli, and the recombinant His-tagged fusion protein was purified and characterized. The gene annotated panE was the sole gene responsible for the reduction of the 2-keto acids derived from leucine, isoleucine, and valine, while ldh, encoding L-lactate dehydrogenase, was responsible for the reduction of the 2-keto acids derived from phenylalanine and methionine. The kinetic parameters of the His-tagged PanE showed the highest catalytic efficiencies with 2-ketoisocaproate, 2-ketomethylvalerate, 2-ketoisovalerate, and benzoylformate (V max /K m ratios of 6,640, 4,180, 3,300, and 2,050 U/mg/mM, respectively), with NADH as the exclusive coenzyme. For the reverse reaction, the enzyme accepted D-2-hydroxyacids but not L-2-hydroxyacids. Although PanE showed the highest degrees of identity to putative NADP-dependent 2-ketopantoate reductases (KPRs), it did not exhibit KPR activity. Sequence homology analysis revealed that, together with the Dmandelate dehydrogenase of Enterococcus faecium and probably other putative KPRs, PanE belongs to a new family of D-2-hydroxyacid dehydrogenases which is unrelated to the well-described D-2-hydroxyisocaproate dehydrogenase family. Its probable physiological role is to regenerate the NAD ؉ necessary to catabolize branched-chain amino acids, leading to the production of ATP and aroma compounds.

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“…It is known that benzoylformate is a favorable substrate for NADdependent D-mandelate dehydrogenases (D-ManDH) that have been purified from L. curvatus (18) and Enterococcus faecalis (36). However, much less information has been ob- (19) exhibit quite similar trends in their substrate specificities, although the former enzyme exhibits still markedly higher activity toward pyruvate and hydroxypyruvate than the latter one ( Fig. 1B and C).…”

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“…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.…”

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

et al. 2003

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, 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...

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Potential of R-2-Hydroxyisocaproate dehydrogenase from Lactobacillus casei for stereospecific reductions

Cited by 48 publications

References 41 publications

Two Forms of NAD-Dependent d -Mandelate Dehydrogenase in Enterococcus faecalis IAM 10071

Two Forms of NAD-Dependent d -Mandelate Dehydrogenase in Enterococcus faecalis IAM 10071

The d -2-Hydroxyacid Dehydrogenase Incorrectly Annotated PanE Is the Sole Reduction System for Branched-Chain 2-Keto Acids in Lactococcus lactis

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

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