Biosynthesis of acetyl-CoA synthase and its derivatives

Acetyl-CoA synthase (a.k.a. carbon monoxide dehydrogenase) from Clostridium thermoaceticum (ACS_Ct/CODH_Ct) is an α₂β₂ tetramer containing three metal-centers called the A-, B-, and C-clusters. This enzyme catalyzes the reversible reduction of CO₂ to CO at the C-cluster, and the synthesis of acetyl-CoA at the A-cluster. Genes acsA and acsB encoding the β and α subunits were cloned into Escherichia coli JM109 and overexpressed at 37°C under anaerobic conditions with Ni supplementation. The resulting recombinant His-tagged protein (AcsAB) exhibited CO oxidation activity and EPR signals with g-values and low spin intensities indistinguishable from those of the reduced states of the B- and C-clusters of ACS_Ct from which Ni had been removed from the A-cluster. Upon overnight exposure to NiCl₂, the resulting recombinant enzyme (ACSEC) developed CO exchange activity and exhibited an EPR signal indistinguishable from the NiFeC signal of Ni-replete ACS_Ct.; Two ORF's, acsF and acsG were identified in the acs operon. The predicted amino acid sequence of acsG has a high percentage of similarity to seven other proteins from archaea and Rhodospirillum rubrum, all of which contain ACS/CODH's. AcsG has a conserved “P-loop” which indicates ATP/GTP binding and is implicated in the C-cluster-Ni-insertion process through comparison to its homolog, CooC from R. rubrum.; Furthermore, a fusion protein of glutathione S-transferase and AcsG that exhibited ATP hydrolysis was isolated.; Three mini-metalloproteins of the acetyl-CoA synthase active site, a 737-, a 436-, and a 223-mer were similarly biosynthesized in E. coli as AcsAB. The 737- and 436-mers each contained a redox-active [Fe ₄S₄]2+/1+ cluster. The Ni-activated 737-mer exhibited an EPR signal and spin intensity indistinguishable from the NiFeC signal of ACS_Ct. Unexpectedly, activated 737-mer also catalyzed the synthesis of acetyl-CoA, though at a rate ∼10-fold slower than ACS _Ct. The Ni-activated 436-mer exhibited an EPR signal indistinguishable from the pNiFeC signal of previously isolated α (Xia & Lindahl, 1996) but was catalytically inactive. The 223-mer contained a redox-inactive Fe₄S₄ cluster and was EPR and catalytically inactive. The characterizations of the three mini-proteins revealed proportional loss of physical properties that characterize the active site as the protein scaffold diminished in size.