Evolutionary history of carbon monoxide dehydrogenase/acetyl-CoA synthase, one of the oldest enzymatic complexes

Abstract

Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) is a five-subunit enzyme complex responsible for the carbonyl branch of the Wood-Ljungdahl (WL) pathway, considered one of the most ancient metabolisms for anaerobic carbon fixation, but its origin and evolutionary history have been unclear. While traditionally associated with methanogens and acetogens, the presence of CODH/ACS homologs has been reported in a large number of uncultured anaerobic lineages. Here, we have carried out an exhaustive phylogenomic study of CODH/ACS in over 6,400 archaeal and bacterial genomes. The identification of complete and likely functional CODH/ACS complexes in these genomes significantly expands its distribution in microbial lineages. The CODH/ACS complex displays astounding conservation and vertical inheritance over geological times. Rare intradomain and interdomain transfer events might tie into important functional transitions, including the acquisition of CODH/ACS in some archaeal methanogens not known to fix carbon, the tinkering of the complex in a clade of model bacterial acetogens, or emergence of archaeal-bacterial hybrid complexes. Once these transfers were clearly identified, our results allowed us to infer the presence of a CODH/ACS complex with at least four subunits in the last universal common ancestor (LUCA). Different scenarios on the possible role of ancestral CODH/ACS are discussed. Despite common assumptions, all are equally compatible with an autotrophic, mixotrophic, or heterotrophic LUCA. Functional characterization of CODH/ACS from a larger spectrum of bacterial and archaeal lineages and detailed evolutionary analysis of the WL methyl branch will help resolve this issue.

Keywords: LUCA; Wood–Ljungdahl pathway; acetogens; evolution; methanogens.

Fig. 1.

(A) The reactions of the WL pathway. In the methyl branch, CO₂ is progressively reduced to formyl/formate (-CHO, HCOOH), methenyl (-CH), methylene (-CH₂), and eventually methyl (-CH₃). There are two nonhomologous versions of the methyl branch, each using a different cofactor to which the reduced carbon compounds are bound, tetrahydromethanopterin (H₄MPT) or tetrahydrofolate (THF), which are commonly associated with methanogenic Archaea and acetogenic Bacteria, respectively. The carbonyl branch reduces a CO₂ molecule to CO (carbonyl moiety), which is combined with the methyl coming from the methyl branch and CoA to form acetyl-CoA. This reaction is achieved through the CODH/ACS enzymatic complex. [H] denotes one reducing equivalent (=1e⁻+1H⁺). (B) Nomenclature and function of the CODH/ACS subunits. (C) Organization of the CODH/ACS cluster in Bacteria and Archaea with indication of homologous subunits. The genes in yellow correspond to the homologous accessory proteins CooC and AcsF, which are responsible for nickel insertion.

Fig. 2.

Distribution of complete or almost complete CODH/ACS complexes mapped on Bayesian reference phylogenies of Archaea (A) and Bacteria (B) based, respectively, on a concatenation of 41 markers (8,710-amino acid positions) (26), and from a concatenation of RNA polymerase subunits B, B′, and IF-2 (2,337-amino acid positions) (this work). Both trees were calculated in PhyloBayes, with the CAT+GTR+Γ4 model. Values at nodes represent Bayesian posterior probabilities. The scale bar represents the average number of substitutions per site. The archaeal and bacterial trees are rooted according to ref. , i.e., in Archaea between two large clades corresponding to cluster I and cluster II, and in Bacteria between two large clades roughly corresponding to Terrabacteria and Gracilicutes. Partial CODH/ACS clusters in one member of Hadesarchaea and one of Thaumarchaeota composed of only two of five subunits (CdhDE and CdhAB, respectively) have been omitted, but are included in Dataset S1. The clades corresponding to uncultured nanosized lineages in both Archaea [Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, Nanohaloarchaeota (DPANN)] and Bacteria [candidate phyla radiation (CPR)] were not included in these reference trees because they do not have CODH/ACS and their placement needs to be assessed with more in-depth analysis. For discussion and details on analyses, see main text and Methods.

Fig. 3.

Bayesian phylogeny based on a concatenation of the five subunits of the archaeal-type CODH/ACS (CdhABCDE, 2,063-aa positions), calculated by PhyloBayes, with the CAT+GTR+Γ4 model. Black diamonds indicate instances of intradomain transfers (see main text for discussion). Cluster I and cluster II refer to the clades identified in the reference archaeal phylogeny in Fig. 2A. Values at nodes are posterior probabilities and bootstrap supports calculated by maximum likelihood in IQTree under the TEST option (100 replicates). Nodes below 0.95 posterior probability support were collapsed. Scale bar represents the average number of substitutions per site. For details on analyses, see Methods.

Fig. 4.

Bayesian phylogeny based on a concatenation of (A) the two oxidoreductase module subunits (CdhAC, 1,248-aa positions) and (B) the three methyltransferase module subunits (AcsE-CdhDE, 965-aa positions) calculated in PhyloBayes, with the CAT+GTR+Γ4 model. Terrabacteria and Gracilicutes refer to the clades identified in the reference bacterial phylogeny in Fig. 2B. Black diamonds indicate instances of intradomain transfers: recent ones from Firmicutes to Acidobacteria, Spirochaetae, and from Deltaproteobacteria to Dehalococcoidia; and three more ancient ones from Chloroflexi (Anaerolineae) to Nitrospirae, and from Aerophobetes/Acetothermia to Plactomycetes/Omnitrophica. The direction of these transfers was deduced by comparing the reference and gene trees to identify misplaced clades (recipients). Values at nodes are posterior probabilities and bootstrap supports calculated by maximum likelihood. Scale bars represent the average number of substitutions per site. For discussion and details on analyses, see main text and Methods.

Fig. 5.

Alternative scenarios for the nature of the CODH/ACS enzyme complex in LUCA and the transition to bacterial and archaeal types in the two domains (see main text for discussion). Inward facing arrows indicate emergence of a new subunit, and outward facing arrows indicate subunit loss.