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Journal of Bacteriology, March 2002, p. 1571-1577, Vol. 184, No. 6
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.6.1571-1577.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Identification of an Acetoacetyl Coenzyme A Synthetase-Dependent Pathway for Utilization of L-(+)-3-Hydroxybutyrate in Sinorhizobium meliloti

Punita Aneja,^1, Renata Dziak,² Guo-Qin Cai,^1, and Trevor C. Charles^1,2*

Department of Natural Resource Sciences, McGill University, Ste-Anne-de-Bellevue, Quebec H9X 3V9 ,¹ Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada²

Received 11 September 2001/ Accepted 4 December 2001

D-(-)-3-Hydroxybutyrate (DHB), the immediate depolymerization product of the intracellular carbon store poly-3-hydroxybutyrate (PHB), is oxidized by the enzyme 3-hydroxybutyrate dehydrogenase to acetoacetate (AA) in the PHB degradation pathway. Externally supplied DHB can serve as a sole source of carbon and energy to support the growth of Sinorhizobium meliloti. In contrast, wild-type S. meliloti is not able to utilize the L-(+) isomer of 3-hydroxybutyrate (LHB) as a sole source of carbon and energy. In this study, we show that overexpression of the S. meliloti acsA2 gene, encoding acetoacetyl coenzyme A (acetoacetyl-CoA) synthetase, confers LHB utilization ability, and this is accompanied by novel LHB-CoA synthetase activity. Kinetics studies with the purified AcsA2 protein confirmed its ability to utilize both AA and LHB as substrates and showed that the affinity of the enzyme for LHB was clearly lower than that for AA. These results thus provide direct evidence for the LHB-CoA synthetase activity of the AcsA2 protein and demonstrate that the LHB utilization pathway in S. meliloti is AcsA2 dependent.

Present address: Department of Biology, McMaster University, Hamilton, Ontario L8S 4K1, Canada.

Present address: BASF Corporation Consumer Products & Life Science Division, Agricultural Products Center, Research Triangle Park, NC 27709.

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