Identification and Characterization of a Re -Citrate Synthase in Dehalococcoides Strain CBDB1

The genome annotations of all sequencedDehalococcoides strains lack a citrate synthase, although physiological experiments have indicated that such an activity should be encoded. We here report that aRe face-specific citrate synthase is synthesized byDehalococcoides strain CBDB1 and that this function is encoded by the gene cbdbA1708 (NCBI accession numberCAI83711 ), previously annotated as encoding homocitrate synthase. Gene cbdbA1708 was heterologously expressed inEscherichia coli , and the recombinant enzyme was purified. The enzyme catalyzed the condensation of oxaloacetate and acetyl coenzyme A (acetyl-CoA) to citrate. The protein did not have homocitrate synthase activity and was inhibited by citrate, and Mn2+ was needed for full activity. The stereospecificity of the heterologously expressed citrate synthase was determined by electrospray ionization liquid chromatography-mass spectrometry (ESI LC/MS). Citrate was synthesized from [2-13 C]acetyl-CoA and oxaloacetate by theDehalococcoides recombinant citrate synthase and then converted to acetate and malate by commercial citrate lyase plus malate dehydrogenase. The formation of unlabeled acetate and13 C-labeled malate proved theRe face-specific activity of the enzyme. Shotgun proteome analyses of cell extracts of strain CBDB1 demonstrated that cbdbA1708 is expressed in strain CBDB1.