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

Abstract

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.

Inactivation of the panE Gene in Lactococcus lactis Enhances Formation of Cheese Aroma Compounds

“…L. lactis IFPL953⌬panE produced higher (P Ͻ 0.05) levels of KIC (derived from leucine) and ␣-ketopropionic acid (pyruvate) than IFPL953 ( Table 2). The accumulation of KIC was related to the absence of HA-DH activity in the mutant strain, since KIC is the preferred substrate for this activity and PanE is the sole enzyme responsible for the reduction of branched-chain ␣-keto acids in L. lactis (6). Growth of IFPL953⌬panE in CDM and milk produced higher levels of 3-methylbutanal and 3-methylbutanol than IFPL953.…”

“…The ␣-keto acids are subsequently degraded either into aldehydes, which are further converted into alcohols, carboxylic acids, and esters, or into ␣-hydroxy acids, which are not aroma compounds (1). Two key enzymes are involved in these reactions, an ␣-ketoacid decarboxylase (KivD) responsible for the transformation of the ␣-keto acids into aldehydes (5) and an ␣-hydroxyacid dehydrogenase (PanE) responsible for the conversion of the ␣-keto acids into the nonaromatic ␣-hydroxy acids (6). Hydroxyacid dehydrogenases (HA-DHs) negatively affect aroma production by competing with decarboxylases for ␣-keto acids (7).…”

“…The mutation was verified by sequencing the 1,645-bp PCR product ⌬panE (Table 1). L. lactis IFPL953⌬panE and the wild-type strain were analyzed for HA-DH activity using ␣-ketoisocaproic acid (KIC) as the substrate (6), showing L. lactis IFPL953 values of 5.78 U/mg (1 U is defined as the quantity of enzyme that oxidizes 1 mol NADH per minute at 37°C), whereas the mutant strain had lost the HA-DH activity. The effect of the mutation on L. lactis IFPL953⌬panE growth was tested by comparing the growth to that of L. lactis IFPL953 grown in a buffered chemically defined medium (CDM) (12) for 24 h (Fig.…”

Enantioselective lactic acid production by an Enterococcus faecium strain showing potential in agro-industrial waste bioconversion: Physiological and proteomic studies

“…A recent study by Chambellon et al (2009) brought evidences that a number of enzymes annotated as ketopantoate reductase on the basis of primary sequence homologies found in LAB, could belong to a new family of proteins, known as 2-hydroxyacid dehydrogenases, involved in amino acid catabolism. Notably, in LAB the first step of amino acid catabolism is generally catalyzed by aminotransferases, which generate 2-oxo acids (Chambellon et al, 2009).…”

“…Notably, in LAB the first step of amino acid catabolism is generally catalyzed by aminotransferases, which generate 2-oxo acids (Chambellon et al, 2009). 2-oxo acids can then be either oxidized by 2-oxo acid dehydrogenases, or reduced by 2-hydroxyacid dehydrogenases ( Fig.…”