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

Hydroxyacid dehydrogenases of lactic acid bacteria, which catalyze the stereospecific reduction of branched-chain 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 d-mandelate 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.Hydroxyacid dehydrogenases catalyze the stereospecific and reversible reduction of 2-keto acids to 2-hydroxyacids. 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. Indeed, overexpression of the d-2-hydroxyisocaproate dehydrogenase (d-2-HicDH) of Lactobacillus casei has been shown previously to decrease the production of aroma compounds and to delay flavor formation in low-fat cheddar cheese (12). Additionally, hydroxyacid dehydrogenases are involved in the biopreservation properties of lactic acid bacteria, because certain 2-hydroxyacids exhibit antifungal and antilisterial activities (15, 37, 64). Several hydroxyacid dehydrogenases in lactic acid bacteria have been characterized previously (21, 27). Lactate dehydrogenases (LDH), responsible for the specific reduction of pyruvate to lactic acid, have been studied extensively (2, 7, 13, 16, 21, 40, 49, 54). HicDHs and mandelate dehydrogenases (manDHs), active toward a broad range of 2-keto acids, including straight-chain aliphatic 2-keto acids, branched-chain 2-keto acids, and 2-keto acids with aromatic side chains, in several lactic acid bacteria have also been characterized previously (5, 6, 27, 28, 29, 38, 39, 50). Although manDHs and HicDHs of lactic acid bacteria prefer 2-ketoisocaproate (KIC) among 2-keto acid substrates, they differ in their activities toward C-3-branched substrates. In particular, manDHs exhibit high levels of activity toward 2-ketoisovalerate (KIV) and benzoylformate, unlike HicDHs. LDHs, HicDHs, and manDHs are divided into two groups, the l and d groups, depending on the stereoisomer produced. d-LDHs and d-HicDHs are members of the same family of 2-hydroxyacid dehydrogenases, which is distinct from the l-LDH family (13, 54). In general, each lactic acid bacterium contains several hydroxyacid dehydrogenases.In Lactococcus lactis, which is widely employed as a starter in cheese production, the main LDH is an l-LDH activated by fructose 1,6-bisphosphate (FBP) (23). In addition to the ldh gene that encodes the l-LDH (40), L. lactis contains other genes showing significant levels of similarity to the hydroxyacid dehydrogenases of other lactic acid bacteria (9), but their functions remain unknown, except for that of the recently identified ldhB gene. ldhB is a silent gene that can be activated in ldh-deficient strains via an IS981 element-specific insertion to produce a functional LDH (10). However L. lactis produces 2-hydroxyacids from the 2-keto acids derived from amino acids not only in vitro, using resting cells, but also in cheese (65, 66). In L. lactis, the catabolism of the aromatic and branched-chain amino acids that are precursors of aroma compounds is initiated by aminotransferases, producing 2-keto acids. These 2-keto acids can be further catabolized into carboxylic acids via a 2-keto acid dehydrogenase, a transacetylase, and a kinase or reduced to 2-hydroxyacids by a hydroxyacid dehydrogenase (66) (Fig. ​(Fig.1).1). In some strains, branched-chain 2-keto acids can also be decarboxylated to aldehydes that are potent aroma compounds. The aim of the present study was to identify and characterize the enzyme(s) involved in the production of 2-hydroxyacids from the 2-keto acids derived from phenylalanine and branched-chain amino acids and to evaluate its impact on amino acid catabolism.Open in a separate windowFIG. 1.Leucine catabolism pathway in L. lactis TIL46. AT, aminotransferase; HADH, hydroxyacid dehydrogenase; TA, transacetylase; KDH, keto acid dehydrogenase; HIC, 2-hydroxyisocaproate.