Published online 22 July 2009Nucleic Acids Research, 2009, Vol. 37, No. 175619–5631
Diversity and dispersal of a ubiquitous protein
family: acyl-CoA dehydrogenases
Yao-Qing Shen*, B. Franz Lang and Gertraud Burger
Robert Cedergren Center for Bioinformatics and Genomics, Biochemistry Department, Universite ´ de Montre ´al,
2900 Edouard-Montpetit, Montreal, QC, H3T 1J4, Canada
Received May 18, 2009; Revised June 17, 2009; Accepted June 18, 2009
Acyl-CoA dehydrogenases (ACADs), which are key
enzymes in fatty acid and amino acid catabolism,
form a large, pan-taxonomic protein family with at
least 13 distinct subfamilies. Yet most reported
ACAD members have no subfamily assigned, and
little is known about the taxonomic distribution
and evolution of the subfamilies. In completely
sequenced genomes from approximately 210 spe-
cies (eukaryotes, bacteria and archaea), we detect
ACAD subfamilies by rigorous ortholog identifica-
tion combining sequence similarity search with
phylogeny. We then construct taxonomic subfam-
trees with orthologous proteins. Subfamily profiles
provide unparalleled insight into the organisms’
energy sources based on genome sequence alone
and further predict enzyme substrate specificity,
thus generating explicit working hypotheses for
targeted biochemical experimentation. Eukaryotic
ACAD subfamilies are traditionally considered as
mitochondrial proteins, but we found evidence that
in fungi one subfamily is located in peroxisomes
and participates in a distinct b-oxidation pathway.
Finally, we discern horizontal transfer, duplication,
loss and secondary acquisition of ACAD genes
during evolution of this family. Through these unor-
thodox expansion strategies, the ACAD family is
proficient in utilizing a large range of fatty acids
shaped the evolutionary history of many other
ancient protein families.
From the last two decades of intensive research especially
in mammals, acyl-CoA dehydrogenases (ACADs) are now
known as a large and biologically important enzyme
family. Genetic defects of the corresponding genes cause
severe health problems in human, including hypoglycemia,
neuromuscular pathology and even death (1). While
ACAD proteins occur in all three domains of life, animals
possess the largest number of distinct subfamilies.
In human, for example, 11 different ACAD enzymes
have been recognized (2–12). These proteins, which in
eukaryotes are localized in mitochondria, share up to
?50% amino acid identity among each other (Table 1)
and catalyze similar biochemical reactions: the oxidation
of diverse acyl-CoA compounds, produced during the deg-
radation of fat and protein, to enoyl-CoA (Figure 1).
ACAD subfamilies are distinguished by the metabolic
pathways in which they participate, and by their substrate
specificity (Figure 1, Table 2). Five subfamilies participate
in b-oxidation of fatty acids, with optimal activity for
acyl-CoA substrates of particular chain length, short
(ACADS), medium (ACADM), long (ACADL), or very
long (ACADV and ACADV2) (11–15). Four other sub-
families are implicated in amino acid degradation. After
removal of the amino groups from isoleucine, leucine,
lysine/trytophan and valine, the remaining branched
acyl-CoA is dehydrogenated by short/branched chain
acyl-CoA dehydrogenase (ACDSB), isovaleryl-CoA dehy-
drogenase (IVD), glutaryl-CoA dehydrogenase (GCDH)
and isobutyryl-CoA dehydrogenase (IBD), respectively
(3,5–7). The most recently identified subfamilies, ACD10
and ACD11, are of yet unknown function (8,9). Two addi-
tional subfamilies have been reported in bacteria: fadE
degrades a broad range of substrates from short to long
chain acyl-CoAs (16,17), while fadE12 prefers medium-
chain length molecules (18). The reaction mechanism
and 3D structure of ACAD enzymes have been reviewed
by others (19,20).
In eukaryotes, b-oxidation involving ACAD enzymes
takes place in mitochondria. Eukaryotes also possess
peroxisomal b-oxidation catalyzed by acyl-CoA oxidase
(ACOX) instead of ACAD proteins. The two families
resemble each other in several aspects. ACOX proteins
share remote yet significant sequence similarity with
ACAD proteins, and also catalyze the conversion of
acyl-CoA to enoyl-CoA. But unlike the ACAD family,
ACOX proteins occur predominantly in eukaryotes, are
*To whom correspondence should be addressed. Tel: +1 514 343 6111 2848; Fax: +1 514 343 2210; Email: email@example.com
? 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
located exclusively in peroxisomes and function by a
re-oxidized by molecular oxygen, generating H2O2(20);
ACAD enzymes, in contrast, having only low reactivity
with molecular oxygen, are re-oxidized by electron-trans-
ferring flavoproteins, which in turn pass the electrons to
the respiratory chain, generating H2O. Insight into the
origin of the ACOX family will critically depend on a
better understanding of the ACAD family, which is the
focus of the study reported here.
ACOX proteins are
Our current knowledge about ACAD proteins is limited
to a few model organisms. There has been no comprehen-
sive survey of ACAD enzymes, except for genome-wide
in silico screens in fungi without subfamily identification
(21,22). Further, it is unclear whether the 11 subfamilies
recognized in human are conserved throughout animals
or even beyond. One reason for these shortcomings is
that in public data repositories, sequences are generally
annotated indistinctively as ‘acyl-CoA dehydrogenase’.
This is because in BLAST searches, remote ACAD
Figure 1. Optimal substrates of ACAD subfamilies. C4, etc., length of the acyl-CoA chain. C16:1, unsaturated fatty acid with one double bond.
Subfamilies in the left part of the figure are involved in fatty acid degradation. Those in the right part are involved in amino acid degradation. ‘R’
represents straight alkyl chain.
Table 1. Pairwise sequence similarities between human ACAD subfamily membersa
ACD11 ACADSACADM ACADLACADV ACADV2ACDSBGCDH IVD IBD fadEfadE12
aAll subfamily members are from human, except for fadE and fadE12, which are prokaryotic subfamilies. Percentage of identical residues in aligned
region by BLAST. Sequences are obtained from SwissProt. Sequence IDs are listed in Table 2.
Nucleic Acids Research, 2009, Vol. 37,No. 17
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