Identification and Characterization of Re-Citrate Synthase in Syntrophus aciditrophicus

• Published in: Journal of Bacteriology 2013-04, Vol.195 (8), p.1689-1696
• Main Authors: Kim, Marie, Le, Huynh, McInerney, Michael J, Buckel, Wolfgang
• Format: Article
• Language: English
• Subjects: acetates; acetyl coenzyme A; Acyltransferases - genetics; Acyltransferases - metabolism; adenosine triphosphate; amino acid sequences; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteriology; Base Sequence; calcium; citrates; Clostridium; Clostridium kluyveri; Deltaproteobacteria - enzymology; DNA, Bacterial - genetics; Enzymes; Escherichia coli; Fatty acids; Fermentation; Gene Expression Regulation, Bacterial - physiology; Gene Expression Regulation, Enzymologic - physiology; gene overexpression; Genes; Genomics; glutamic acid; Glutamic Acid - genetics; Glutamic Acid - metabolism; Gram-negative bacteria; magnesium; Microorganisms; potassium; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA, Bacterial - genetics; RNA, Bacterial - metabolism; sequence analysis; Substrate Specificity; Syntrophus; Syntrophus aciditrophicus
• ISSN: 0021-9193; 1098-5530; 1067-8832
• DOI: 10.1128/JB.02185-12

Glutamate is usually synthesized from acetyl coenzyme A (acetyl-CoA) via citrate, isocitrate, and 2-oxoglutarate. Genome analysis revealed that in Syntrophus aciditrophicus, the gene for Si-citrate synthase is lacking. An alternative pathway starting from the catabolic intermediate glutaconyl-CoA via 2-hydroxyglutarate could be excluded by genomic analysis. On the other hand, a putative gene (SYN_02536; NCBI gene accession no. CP000252.1) annotated as coding for isopropylmalate/citramalate/homocitrate synthase has been shown to share 49% deduced amino acid sequence identity with the gene encoding Re-citrate synthase of Clostridium kluyveri. We cloned and overexpressed this gene in Escherichia coli together with the genes encoding the chaperone GroEL. The recombinant homotetrameric enzyme with a C-terminal Strep-tag (4 × 72,892 Da) was separated from GroEL on a Strep-Tactin column by incubation with ATP, K+, and Mg2+. The pure Re-citrate synthase used only acetyl-CoA and oxaloacetate as the substrates. As isolated, the enzyme contained stoichiometric amounts of Ca2+ (0.9 Ca/73 kDa) but achieved higher specific activities in the presence of Mn2+ (1.2 U/mg) or Co2+ (2.0 U/mg). To determine the stereospecificity of the enzyme, [14C]citrate was enzymatically synthesized from oxaloacetate and [1-14C]acetyl-CoA; the subsequent cleavage by Si-citrate lyase yielded unlabeled acetate and labeled oxaloacetate, demonstrating that the enzyme is a Re-citrate synthase. The production of Re-citrate synthase by S. aciditrophicus grown axenically on crotonate was revealed by synthesis of [14C]citrate in a cell extract followed by stereochemical analysis. This result was supported by detection of transcripts of the Re-citrate synthase gene in axenic as well as in syntrophic cultures using quantitative reverse transcriptase PCR (qRT-PCR).