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Identification and characterization of re-citrate synthase in Syntrophus aciditrophicus.

ABSTRACT: 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 Mg(2+). The pure Re-citrate synthase used only acetyl-CoA and oxaloacetate as the substrates. As isolated, the enzyme contained stoichiometric amounts of Ca(2+) (0.9 Ca/73 kDa) but achieved higher specific activities in the presence of Mn(2+) (1.2 U/mg) or Co(2+) (2.0 U/mg). To determine the stereospecificity of the enzyme, [(14)C]citrate was enzymatically synthesized from oxaloacetate and [1-(14)C]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 [(14)C]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).

SUBMITTER: Kim M

PROVIDER: S-EPMC3624545 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Identification and characterization of re-citrate synthase in Syntrophus aciditrophicus.

Kim Marie M Le Huynh H McInerney Michael J MJ Buckel Wolfgang W

Journal of bacteriology 20130201 8

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/homoc ...[more]

PMID: 23378508

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Project description:Syntrophus aciditrophicus is a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation by S. aciditrophicus However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome of S. aciditrophicus leaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA) and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show that S. aciditrophicus uses AMP-forming, acetyl-CoA synthetase (Acs1) for ATP synthesis from acetyl-CoA. acs1 mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, of S. aciditrophicus grown in pure culture and coculture. Cell extracts of S. aciditrophicus had low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified from S. aciditrophicus and recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4) in S. aciditrophicus cells support the operation of Acs1 in the acetate-forming direction. Thus, S. aciditrophicus has a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase. Bacteria use two enzymes, phosphate acetyltransferase and acetate kinase, to make ATP from acetyl-CoA, while acetate-forming archaea use a single enzyme, an ADP-forming, acetyl-CoA synthetase, to synthesize ATP and acetate from acetyl-CoA. Syntrophus aciditrophicus apparently relies on a different approach to conserve energy during acetyl-CoA metabolism, as its genome does not have homologs to the genes for phosphate acetyltransferase and acetate kinase. Here, we show that S. aciditrophicus uses an alternative approach, an AMP-forming, acetyl-CoA synthetase, to make ATP from acetyl-CoA. AMP-forming, acetyl-CoA synthetases were previously thought to function only in the activation of acetate to acetyl-CoA.

Dataset Information

Identification and characterization of re-citrate synthase in Syntrophus aciditrophicus.

Publications

Identification and characterization of re-citrate synthase in Syntrophus aciditrophicus.

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