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

Abstract

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.Poly-3-hydroxybutyrate (PHB), a bacterial intracellular reserve of carbon and reducing energy, accumulates when a nutrient other than carbon is limiting for growth, as reviewed by Madison and Huisman (18). Based on biochemical evidence, the metabolism of PHB has been proposed to be a cyclical process, comprising the pathways for synthesis and degradation of PHB. Synthesis of PHB occurs when excess carbon, in the form of acetyl coenzyme A (acetyl-CoA), is condensed via a ketothiolase (EC 2.3

Characterization ofbdhA, encoding the enzyme d-3-hydroxybutyrate dehydrogenase, fromSinorhizobiumsp. strain NGR234

“…We have previously shown that the presence of multiple copies of the S. meliloti acsA2 gene could suppress the 3-hydroxybutyrate utilization phenotype of the S. meliloti bdhA mutant [26]. To test whether a similar phenomenon operates in NGR234, plasmid pGQ105 [27], bearing the S. meliloti acsA2 gene on an IncP plasmid, was introduced into NGRPA2.…”

Multi-dimensional Roles of Ketone Bodies in Fuel Metabolism, Signaling, and Therapeutics

“…Ketone bodies are a vital alternative metabolic fuel source for all the domains of life, eukarya, bacteria, and archaea (Aneja et al, 2002; Cahill GF Jr, 2006; Krishnakumar et al, 2008). Ketone body metabolism in humans has been leveraged to fuel the brain during episodic periods of nutrient deprivation.…”

Ketone body metabolism and cardiovascular disease

“…Metabolism of ketone bodies is conserved among eukarya, bacteria, and archaea (8,33,113). Ketone bodies are synthesized in liver from acetyl-CoA derived primarily from fatty acid oxidation and are transported to extrahepatic tissues for terminal oxidation during physiological states characterized by limited carbohydrate and surplus fatty acid availability [reviewed in (137,167); Fig.…”