Novel Domain Arrangement in the Crystal Structure of a Truncated Acetyl-CoA Synthase from Moorella thermoacetica^,

Abstract

Ni-dependent Acetyl-CoA synthase (ACS) and CO dehydrogenase (CODH) constitute the central enzyme complex of the Wood-Ljungdahl pathway of acetyl-CoA formation. The crystal structure of a recombinant bacterial ACS lacking the N-terminal domain that interacts with CODH shows a large reorganization of the remaining two globular domains, producing a narrow cleft of suitable size, shape and nature to bind CoA. Sequence comparisons with homologous archaeal enzymes that naturally lack the N-terminal domain show that many amino acids lining this cleft are conserved. Besides the typical [4Fe-4S] center, the A-cluster contains only one proximal metal ion that, according to anomalous scattering data, is most likely Cu or Zn. Incorporation of a functional Ni 2 Fe 4 S 4 A-cluster would require only minor structural rearrangements. Using available structures, a plausible model of the interaction between CODH and the smaller ACS in archaeal multi-enzyme complexes is presented, along with a discussion of evolutionary relationships of the archaeal and bacterial enzymes.Ni-dependent acetyl coenzyme A synthase (ACS) is a NiFeS-cluster enzyme that requires strict anoxic conditions to function. In the acetogenic bacterium Moorella thermoacetica (Mot), the ACS α-subunit forms an α 2 β 2 heterotetramer with the Ni-containing carbon monoxide dehydrogenase (CODH) β-subunit (1). This association constitutes the central enzyme complex of the anaerobic Wood-Ljungdahl pathway of acetyl-CoA synthesis from CO 2 (2,3). The structure of this complex is known in two crystal forms (4,5) and shows a long hydrophobic tunnel network that allows the CO produced at the CODH active site, the C-cluster, to be used at the ACS active site, the A-cluster, without being released to the medium. Our understanding of CO 2 reduction to CO by CODH has progressed significantly thanks to the recent crystal structures of key catalytic intermediates in the homodimeric β 2 enzyme from † This study was supported by institutional funding from the CEA and the CNRS and by the National Institute of Health (GM046441 PAL). ‡ The atomic coordinates and structure factors (accession number 3GIT) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics (http://www.pdb.org).*To whom correspondence should be addressed. AV: Tel. +33438789606, Fax +33438785122, E-mail anne.volbeda@ibs.fr; JCFC: Tel. +33438785920, Fax +33438785122, E-mail juan.fontecilla@ibs.fr.. # Current address: Department of Chemistry, Institute of Biomedical Sciences, Fudan University, Shanghai, 200043, P.R. China Supporting Information Available. Superposition statistics of bacterial CODH/ACS and archaeal CODH, sequence alignment of bacterial ACS with archaeal CODH and structure comparison of CODH and HCP. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 August 25. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptC...

Tight Coupling of Partial Reactions in the Acetyl-CoA Decarbonylase/Synthase (ACDS) Multienzyme Complex from Methanosarcina thermophila

“…Interestingly, the A cluster activities of the proteins correlated with the type of signal observed, in that only samples that displayed type I signals (ACS Ch and the ACDS complex) were able to carry out significant carbonyl exchange activity. Recent crystallographic analyses on an N-terminally truncated form of ACS from M. thermoacetica indicated that a substantial reorientation of the relative positions of the central and C-terminal domains had taken place, as compared with either the closed or open forms of the protein where some degree of contact with the N-terminal region is always maintained (50). Thus, the change in the EPR signal from type I to type II is seen to take place in parallel with the conformational changes that result from loss of interactions of the N-terminal domain with the central and C-terminal domains in ACS or in the case of the ACDS complex with the loss of contact of the ␤ subunit with other protein components in the complex such as the ␣ 2 ⑀ 2 CO dehydrogenase subcomponent.…”

“…Of course, those residues are absent in the isolated ␤ subunit, but the possible locations of functionally equivalent residues in the ACDS complex have been postulated from a recent structural alignment analysis of the ACS Mt N-terminal domain with regions of the archaeal ␣ subunit from the M. barkeri ␣ 2 ⑀ 2 enzyme (50), and a sufficiently high structural similarity was found for the authors to propose a model of the region of the CODH ␣ subunit that may interact with the ␤ subunit. In that model, conserved or invariant residues could provide 31 of the total 41 contacts made within 3.8 Å between the N-and the C-terminal domains in the closed form (50). Two residues in the ACS Mt N-terminal domain, Ile-146 and Val-149 (which would correspond to M. barkeri CODH ␣ subunit Leu-507 and Val-510), are in particularly close proximity to the A cluster.…”

Ligand binding at the A-cluster in full-length or truncated acetyl-CoA synthase studied by X-ray absorption spectroscopy

“…According to the paramagnetic mechanism, CO accesses the A-cluster via a channel [ 16 ] and can bind to Ni p to form a Ni(I)-CO species, which upon reduction accepts an additional methyl group, so that subsequent C-C bond formation and S-C coupling with a CoA molecule finally yields acetyl-CoA [ 6 , 14 , 21 – 24 ]. The influence of the N-terminal and middle ACS domains on the function of the A-cluster has remained a matter of debate [ 14 , 17 , 25 , 26 ]. Interestingly, acetyl-CoA synthesis activity of ACS under in vitro conditions depends on the relative CO concentration.…”

The Conversion of Nickel‐Bound CO into an Acetyl Thioester: Organometallic Chemistry Relevant to the Acetyl Coenzyme A Synthase Active Site

“…[1,4,[8][9][10] In the meantime, a consensus has been reached that HSCoA is the last substrate to bind. [10,11] It is also widely accepted that the resting state contains both Ni centers in the oxidation state +II, and that Ni d mainly serves the stabilization of the structure. Reduction of Ni p , either to Ni I or even to Ni 0 , produces the active state that is capable of creating the acetyl moiety from CO and a methyl species.…”