Exploring the biosynthetic pathways of glutamate and benzoate in Syntrophus aciditrophicus

Syntrophus aciditrophicus thrives syntrophically on benzoate and axenically on crotonate, which is oxidized to acetate and reduced to cyclohexane carboxylate and some benzoate. Genomic, proteomic, and metabolic analyses suggested that degradation and synthesis of benzoate use the same pathway, whereby glutaconyl-CoA serves as central intermediate. In strictly anaerobic bacteria, glutamate is usually synthesized from two acetyl-CoA via pyruvate, oxaloacetate, citrate, and 2-oxoglutarate. As no gene for Si-citrate synthase has been detected in the genome of S. aciditrophicus, we speculated that glutaconyl-CoA via 2-hydroxyglutarate could be the precursor of 2-oxoglutarate for glutamate biosynthesis. Recently, the gene rcs, which is annotated as isopropylmalate/citratemalate/homocitrate synthase in S. aciditrophicus, has been shown to exhibit 49% sequence identity with that coding for Re-citrate from Clostridium kluyveri. We cloned rcs and overproduced the recombinant protein in Escherichia coli. The enzyme was purified aerobically and characterized biochemically as Re-citrate synthase. The highest achieved specific activity was 1.6 U/mg using oxaloacetate and acetyl-CoA as substrates in the presence of Co2+. Pyruvate, 2-oxoglutarate and 2-oxoisovalerate could not replace oxaloacetate; with propionyl-CoA also no activity was observed. No metal was detected in the recombinant protein. Besides Co2+ also Mn2+ stimulates the activity and stabilizes the enzyme. Sulfhydryl reagents partially inactivate the enzyme, but a cysteine residue seems not to be involved in the catalytic site. With [2H3]acetyl-CoA a low intermolecular deuterium isotope effect (kH/kD = 1.4) was measured. Preliminary native PAGE data indicate a homodimeric structure of the enzyme. Labeled glutamate and aspartate were extracted from S. aciditrophicus cells grown on unlabeled crotonate with [1-14C]acetate or 13CO2 and analyzed by oxidative decarboxylation and its radioactivity or by 13C-NMR, respectively. Together with GC-MS data from the universities of Oklahoma and Washington using [1-13C]acetate, the present results support the idea that Re-citrate synthase participates in glutamate biosynthesis, although an incomplete equilibration between labeled acetate and unlabeled crotonate must be considered. Unfortunately, the labeling pattern of glutamate derived from acetate via pyruvate, oxaloacetate and citrate cannot solely be distinguished from that via glutaconyl-CoA and 2-hydroxyglutarate. To study the proposed reversibility of the energy conserving glutaconyl-CoA decarboxylase (Gcd), especially whether the carboxylation of crotonyl-CoA is driven by an electrochemical Na+ gradient, we cloned the genes gcdA, gcdB, and gcdC detected in the genome of S. aciditrophicus. The deduced amino acid sequences show 52%, 51%, 46% and 42% identity to GcdA, B, C1 and C2 from Clostridium symbiosum, respectively, though the (A+P) rich domain of GcdC is missing and a gene for GcdD could not be detected. The S. aciditrophicus genes were expressed individually and in the combinations of gcdAC and gcdABC in E. coli, whereby only the productions of GcdA, GcdC, and GcdAC were successful. GcdA was characterized as carboxytranserase (2 mU/mg with 5 mM D-biotin as artificial acceptor). Purification of the decarboxylase complex by avidin affinity chromatography from S. aciditrophicus cells, grown in a fermenter in Leipzig, was not successful. To uncover the mechanism of transferring Na+ and CO2 in Gcd, a systematic approach of membrane protein overproduction and crystallization should be attempted. Perhaps the lack of the aggregate-forming (A+P) rich domain of GcdC facilitates crystallization.