Acyl-CoA dehydrogenases: Dynamic history of protein family evolution

Abstract

The acyl-CoA dehydrogenases (ACADs) are enzymes that catalyze the alpha,beta-dehydrogenation of acyl-CoA esters in fatty acid and amino acid catabolism. Eleven ACADs are now recognized in the sequenced human genome, and several homologs have been reported from bacteria, fungi, plants, and nematodes. We performed a systematic comparative genomic study, integrating homology searches with methods of phylogenetic reconstruction, to investigate the evolutionary history of this family. Sequence analyses indicate origin of the family in the common ancestor of Archaea, Bacteria, and Eukaryota, illustrating its essential role in the metabolism of early life. At least three ACADs were already present at that time: ancestral glutaryl-CoA dehydrogenase (GCD), isovaleryl-CoA dehydrogenase (IVD), and ACAD10/11. Two gene duplications were unique to the eukaryotic domain: one resulted in the VLCAD and ACAD9 paralogs and another in the ACAD10 and ACAD11 paralogs. The overall patchy distribution of specific ACADs across the tree of life is the result of dynamic evolution that includes numerous rounds of gene duplication and secondary losses, interdomain lateral gene transfer events, alteration of cellular localization, and evolution of novel proteins by domain acquisition. Our finding that eukaryotic ACAD species are more closely related to bacterial ACADs is consistent with endosymbiotic origin of ACADs in eukaryotes and further supported by the localization of all nine previously studied ACADs in mitochondria.

Fig. 1

Maximum likelihood phylogeny of acyl-CoA dehydrogenase protein family. Maximum likelihood phylogeny was estimated from a dataset of 353 amino acid positions from 205 sequences using PHYML (Guindon and Gascuel 2003). Tree was rooted (R) with ACOX and aidB homologs (not shown). Numbers at internodes refer to maximum likelihood bootstrap values. Only bootstrap values of >50% are shown. Gray bar shows the basal tree topology that differed in various analyses and exhibited low bootstrap support (<50%). Thick branches represent well-supported clades corresponding to human ACAD species (marked by circles) that were consistently recovered in all analyses performed. Arrows point to human ACAD homologs. Sequence accession numbers are in parentheses

Fig. 1

Maximum likelihood phylogeny of acyl-CoA dehydrogenase protein family. Maximum likelihood phylogeny was estimated from a dataset of 353 amino acid positions from 205 sequences using PHYML (Guindon and Gascuel 2003). Tree was rooted (R) with ACOX and aidB homologs (not shown). Numbers at internodes refer to maximum likelihood bootstrap values. Only bootstrap values of >50% are shown. Gray bar shows the basal tree topology that differed in various analyses and exhibited low bootstrap support (<50%). Thick branches represent well-supported clades corresponding to human ACAD species (marked by circles) that were consistently recovered in all analyses performed. Arrows point to human ACAD homologs. Sequence accession numbers are in parentheses

Fig. 2

Bayesian phylogeny of glutaryl-CoA dehydrogenase (a) and ACAD10/11 (b) protein subfamilies. Unrooted phylograms were estimated from 377 and 402 amino acids, respectively. Numbers at internodes show posterior probabilities for Bayesian consensus tree. Bars (right to a) refer to clades or groups: A = archaeal taxa, B1–B3 = bacterial taxa, P and E = eukaryotic taxa

Fig. 3

Inferred order of events in evolution of ACAD10/11 subfamily. Bars represent protein domains: long bar = ACAD, medium bar = APH, and short bar = HAD domains. The protein subfamily originated from an ancestral single-domain ACAD10/11, as exemplified in bacterial genomes. In eukaryotes, this single-domain ACAD10/11 acquired a new domain, the APH, and became a two-domain protein as exhibited in plant genomes. Later, most likely before diversification of major nonplant eukaryotic lineages, a duplication event resulted in two paralogs, the ACAD10 and ACAD11. ACAD10 acquired a second domain, the HAD. The single-domain homolog in fungi is most likely the result of an LGT from a bacterial donor that resulted in orthologous replacement of the ancestral ACAD10/11 homolog

Fig. 4

Sequence logo of the last triplet of C-terminal amino acids of eukaryotic ACAD10 and ACAD11 homologs. The data used to generate this terminal motive contained 26 metazoan sequences: 3 plant, 6 fungi, 1 diatom, 1 ciliate, 2 cnidarian, 1 nematode, and 12 vertebrate sequences. The abundance of a residue is directly related to its size. The logo was generated by the weblogo server at http://weblogo.berkeley.edu/using default settings (Crooks et al. 2004)

Fig. 5

Bayesian phylogeny of isovaleryl (a), very long-chain and ACAD9 (b), long-chain (c), and isobutyryl-CoA (d) dehydrogenase subfamilies. Unrooted phylograms were estimated from 384, 544, 375, and 373 amino acids, respectively. Numbers at internodes show posterior probabilities for Bayesian consensus tree. Small triangle in b refers to gene duplication event

Fig. 6

Bayesian phylogeny of short/branched-chain (a), short-chain (b), and medium-chain (c) acyl-CoA dehydrogenase subfamilies. Unrooted phylograms were estimated from datasets of 377, 379, and 376 amino acids, respectively. Numbers at internodes show posterior probabilities for Bayesian consensus tree. Small circles refer to gene duplication events

Fig. 7

Conserved region of the C-terminal extension of VLCAD and ACAD9 exhibiting similarity to ACAD domain (e = 0.001). Alignment of 9 bacterial and 23 eukaryotic VLCAD and ACAD9 homologs. Conserved residues are shaded. Arrows show residues that are invariant or highly conserved across the 32 sequences compared. While Q522 is involved in binding the adenine moiety of the FAD, R544 forms a salt bridge with the invariant E354, apparently supporting the tertiary structure integrity. The K513, W566, and E569 seem to stabilize the tertiary structure as well. In the tetrameric ACADs, interactions across the dimer–dimer interface help stabilize the protein quaternary structure