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.
Chambellon
Emilie
E
INRA, UR 477 Biochimie Bactérienne, F-78350 Jouy-en-Josas, France.
Rijnen
Liesbeth
L
Lorquet
Frédérique
F
Gitton
Christophe
C
van Hylckama Vlieg
Johan E T
JE
Wouters
Jeroen A
JA
Yvon
Mireille
M
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D007651:Keto Acids / Q000378:metabolism*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D013294:Lactococcus lactis / Q000235:genetics / Q000378:metabolism*; D007930:Leucine / Q000378:metabolism; D008954:Models, Biological; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D013379:Substrate Specificity
D001426:Bacterial Proteins; D007651:Keto Acids; D011994:Recombinant Proteins; D010088:Oxidoreductases; D007770:L-Lactate Dehydrogenase; D007930:Leucine