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Journal of Bacteriology, February 2009, p. 873-881, Vol. 191, No. 3
0021-9193/09/$08.00+0     doi:10.1128/JB.01114-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

The D-2-Hydroxyacid Dehydrogenase Incorrectly Annotated PanE Is the Sole Reduction System for Branched-Chain 2-Keto Acids in Lactococcus lactis{triangledown} ,{dagger}

Emilie Chambellon,1 Liesbeth Rijnen,2,{ddagger} Frédérique Lorquet,1,§ Christophe Gitton,1 Johan E. T. van Hylckama Vlieg,2 Jeroen A. Wouters,2 and Mireille Yvon1*

INRA, UR 477 Biochimie Bactérienne, F-78350 Jouy-en-Josas, France,1 Department of Flavour, NIZO Food Research B.V., 6710 BA Ede, The Netherlands2

Received 8 August 2008/ Accepted 20 November 2008

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 (Vmax/Km 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.

* Corresponding author. Mailing address: INRA, Unité de Biochimie Bactérienne, UR 477, 78350 Jouy-en-Josas, France. Phone: 33 1 34 65 21 59. Fax: 33 1 34 65 21 63. E-mail: mireille.yvon{at}jouy.inra.fr

{triangledown} Published ahead of print on 1 December 2008.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

{ddagger} Present address: 66 Chemin du Brias, 30350 Lézan, France.

§ Present address: 165 Avenue Jean-Baptiste Clément, 92140 Clamart, France.

Journal of Bacteriology, February 2009, p. 873-881, Vol. 191, No. 3
0021-9193/09/$08.00+0     doi:10.1128/JB.01114-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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