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Journal of Bacteriology, June 2000, p. 3239-3246, Vol. 182, No. 11
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Branched-Chain -Keto Acid Catabolism via the Gene Products of the bkd Operon in Enterococcus faecalis: a New, Secreted Metabolite Serving as a Temporary Redox Sink

Donald E. Ward,^1, Coen C. van der Weijden,² Marthinus J. van der Merwe,³ Hans V. Westerhoff,² Al Claiborne,¹ and Jacky L. Snoep^2,3,*

Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, North Carolina 27157¹; Department of Molecular Cell Physiology, BioCentrum Amsterdam, Vrije Universiteit, Amsterdam, The Netherlands²; and Department of Biochemistry, University of Stellenbosch, Stellenbosch, South Africa³

Received 21 December 1999/Accepted 28 February 2000

Recently the bkd gene cluster from Enterococcus faecalis was sequenced, and it was shown that the gene products constitute a pathway for the catabolism of branched-chain -keto acids. We have now investigated the regulation and physiological role of this pathway. Primer extension analysis identified the presence of a single promoter upstream of the bkd gene cluster. Furthermore, a putative catabolite-responsive element was identified in the promoter region, indicative of catabolite repression. Consistent with this was the observation that expression of the bkd gene cluster is repressed in the presence of glucose, fructose, and lactose. It is proposed that the conversion of the branched-chain -keto acids to the corresponding free acids results in the formation of ATP via substrate level phosphorylation. The utilization of the -keto acids resulted in a marked increase of biomass, equivalent to a net production of 0.5 mol of ATP per mol of -keto acid metabolized. The pathway was active under aerobic as well as anaerobic conditions. However, under anaerobic conditions the presence of a suitable electron acceptor to regenerate NAD⁺ from the NADH produced by the branched-chain -keto acid dehydrogenase complex was required for complete conversion of -ketoisocaproate. Interestingly, during the conversion of the branched-chain -keto acids an intermediate was always detected extracellularly. With -ketoisocaproic acid as the substrate this intermediate was tentatively identified as 1,1-dihydroxy-4-methyl-2-pentanone. This reduced form of -ketoisocaproic acid was found to serve as a temporary redox sink.

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^* Corresponding author. Mailing address: Department of Biochemistry, University of Stellenbosch, Private Bag X1, Matieland 7602, Stellenbosch, South Africa. Phone: 27-21-808-5844. Fax: 27 21 808 3022. E-mail: jls{at}maties.sun.ac.za.

Present address: Department of Microbiology, Wageningen Agricultural University, Wageningen, The Netherlands.

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Journal of Bacteriology, June 2000, p. 3239-3246, Vol. 182, No. 11
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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