acyl - coa thioesterases ( acots ) represent a group of enzymes that metabolise acyl - coa esters to their corresponding non - esterified fatty acid and coenzyme a ( coash ) .
in addition to ' acyl - coa thioesterases ' , these enzymes also have been described as ' acyl - coa hydrolases ' , ' acyl - coa thioester hydrolases ' and ' deacylases ' .
the reaction catalysed by acot enzymes is very important during fatty acid metabolism . as such
acyl - coa thioesterase expression is widespread and activity is detectable within many tissues and cell types .
acot activity helps to regulate cellular pools , as well as proper ratios , of activated fatty acyl - coas ( acyl - coa esters ) , free fatty acids ( non - esterified fatty acids ) and coash .
these enzymes are found within a number of subcellular locations , including peroxisomes , mitochondria and the cytosol .
peroxisomal and mitochondrial acots are thought to play a major role in fatty acid degradation via -oxidation , which provides energy through the citric acid cycle , as well as in ketone body formation . in order for a fatty acid to be transported into the peroxisome for -oxidation
, it must first be activated to its coenzyme a ester by acyl - coa synthetases .
some acyl - coa esters undergo -oxidation much more slowly than others -- which can effectively sequester large amounts of coash and limit the activity of acyl - coa synthetases and the rate of subsequent fatty acid -oxidation . under these conditions
, acots may act to increase the levels of free coash , thereby restoring fatty acid -oxidation .
mammalian acots are capable of metabolising a wide variety of compounds , including short- , medium- and long - chain acyl - coas .
they can also metabolise polyunsaturated fatty acyl - coas , branched - chain acyl - coas and aromatic - acyl - coas .
these compounds are not only utilised during -oxidation and fatty acid degradation , but also have roles in inflammation , ion channel opening , signal transduction , lipid biosynthesis and gene regulation .
ten human acot genes have been identified to date , compared with 13 in mice and 12 in rats .
these genes can be divided into two groups , based upon differences in sequence and structure .
these enzymes are members of the /-hydrolase fold enzyme superfamily , which includes other esterase - activity - exhibiting enzymes such as carboxylesterases and lipases .
type ii acots make up the second group ; these enzymes are members of the ' hot dog ' fold enzyme superfamily , which includes a wide variety of functionally diverse proteins . despite catalysing the same reaction , type i and type ii enzymes share no similarity in terms of structure or sequence
this observation is reminiscent of the cytochrome p450 ( cyp ) and nitric oxide synthase ( nos ) gene families .
studies with purified nos proteins revealed that they possess p450-like activity ; however , detailed alignment analyses showed < 15 per cent amino acid identity , thereby confirming that these two gene families were not evolutionarily related .
it was thus concluded that this was an example of convergent evolution . in other words , during the evolution of enzyme activities needed at a particular subcellular location ( probably more than 1 billion years ago ) mother nature needed a ' p450-like activity ' and assigned that task to the nearest protein , which happened to be an nos .
the revised nomenclature for human , mouse and rat acyl - coa thioesterase genes was established by hunt et al . and has been accepted by the hugo gene nomenclature committee . in recent years , a number of human acots have been recombinantly expressed and enzymatically characterised , although the exact physiological function and importance of many of these enzymes remain unknown .
to date , a total of ten genes have been identified within the human genome , all of which exhibit acyl - coa thioesterase activity ( table 1 ) .
these ten genes can be sub - divided into two distinct groups , type i and type ii , based on sequence and structural homology .
type i acots comprise the smaller group , which contains only four genes : acot1 , acot2 , acot4 and acot6 .
the larger group includes six genes , and these members are referred to as type ii .
there is no sequence homology between the two groups , and the translated proteins have very different domain architecture , indicating two distinct , evolutionarily unrelated , origins ( figure 1 ) and , most likely , an example of convergent evolution .
list of all human ( acot ) with full gene name , aliases , chromosomal location , isoforms , ncbi refseq mrna accession number , ncbi refseq protein accession number and total amino acid number ( aa # ) domain organisation of human type i and type ii acyl - coa thioesterases ( acots ) .
abbreviations : acth , acyl - coa thioester hydrolase domain ; baat , bile acid coa : amino acid n - acyltransferase ; el , esterase - lipase domain ; hd , ' hot dog ' fold domain ; start , steroidogenic acute regulatory protein ( star)-related lipid transfer ( start ) domain ; them , thioesterase superfamily member .
the type i proteins , acot1 , acot2 , acot4 and acot6 , share a high degree of sequence homology ( table 2 ) .
acot1 and acot2 are the most closely related , having 98 per cent amino acid sequence identity .
acot6 is the least homologous , but still shares 54 - 57 per cent sequence identity with the other members .
the four type i genes form an 80 kilobase gene cluster on chromosome 14q24.3 , indicating that they most likely have arisen as the result of gene duplication .
amino acid sequence identity between type i and type ii human acyl - coa thioesterases ( acots ) .
homology was considered not significant and left blank if the observed identity was less than 15 per cent abbreviations : baat , bile acid coa : amino acid n - acyltransferase ; them , thioesterase superfamily member a similar situation is observed within the mouse and rat orthologues acot1 , acot2 , acot3 , acot4 , acot5 and acot6 , which are clustered on chromosomes 12 d3 and 6q31 , respectively . a cladogram depicting the evolutionary relationships observed between human , mouse and rat type i proteins is shown in figure 2a .
this cladogram clearly shows that , prior to the mammalian radiation approximately 75 million years ago ( mya ) , there was a single ancestral acot1 ( or -2 ) gene ; after the mammalian radiation but before the mouse - rat split at approximately 15 mya , this single ancestor divided twice to become four rodent acot functional genes ( now named acot1 , acot3 , acot4 and acot5 ) . by contrast , after the mammalian radiation , the human single ancestor duplicated into a second functioning gene , leading to acot1 and acot2 . on the other hand , acot4 and acot6 existed before the mammalian radiation and
cladograms depicting the evolutionary relationships between human , mouse ( m ) and rat ( r ) orthologous protein sequences for type i ( a ) and type ii ( b ) acots .
human carboxylesterase 1 ( ces1 ) is an unrelated esterase that was added simply to ' ground ' the tree . in the mouse , the acot3 , acot4 and acot5 enzymes each exhibit specific expression patterns and enzyme substrate specificity .
the lack of a human orthologue to mouse acot3 and acot5 was suggested to be the result of convergent evolution ; it was hypothesised that convergent , functional evolution led to a reduced need for multiple enzymes in humans .
the protein basic local alignment search tool ( blastp ) analysis of all non - redundant protein sequences , however , failed to identify orthologues for acot3 or acot5 in non - rodent species .
this finding suggests that acot4 was an ancestral gene encoding an acot4 enzyme with broad substrate recognition , and that gene duplication events led to acot1 ( or -2 ) , and that further duplication events led to the mouse acot3 and acot5 genes , encoding the acot3 and acot5 enzymes , which have similar substrate specificity to that of mouse acot4 and human acot4 .
analysis of the genomic acot6 sequence indicates transcription of a complete mrna containing three exons ; however , a full - length mrna is not detectable .
subsequent translation begins at a methionine located within the second exon , producing a truncated protein that lacks the n - terminal acyl - coa thioester hydrolase domain found within other type i proteins .
the truncated protein contains all residues required for catalysis and could therefore still produce an active acyl - coa thioesterase .
the blast results indicate that bile acid coa : amino acid n - acyltransferase ( baat ) is the only enzyme to share significant sequence homology with type i acots .
baat is known to participate in the transfer of bile acids from the acyl - coa thioester to either glycine or taurine , which are used for bile salt secretion .
baat contains the same domain architecture and acot1 , acot2 and acot4 ; however , the catalytic triad found within the esterase - lipase domain contains a cysteine , instead of the highly conserved serine residue .
this substitution may explain the difference in activity ( ie transferase versus hydrolase ) observed between the two enzymes . despite sharing catalytic domains , other /-hydrolase fold enzyme superfamily esterases , such as carboxylesterase 1 ( ces1 ) and acetylcholinesterase ( ache ) , share very little sequence homology with their acyl - coa thioesterase relatives .
ces1 has thus been added as an evolutionarily unrelated gene to figure 2a simply to ' ground ' the dendrogram .
none of the type ii proteins shares more than 25 per cent identity and many do not share any significant sequence similarity ( table 2 ) . unlike the type
i genes , type ii acots are widely distributed throughout the genome . with the exception of acot13 ,
all type ii proteins contain double ' hot dog ' domains , which may have evolved as a gene duplication event that allowed for the accommodation of bulky substrates .
acot7 shares 19 per cent identity with acot9 , 22 per cent with acot11 and 24 per cent identity with acot12 .
acot11 and acot12 share 51 per cent sequence identity and both enzymes contain a c - terminal steroidogenic acute regulatory protein ( star)-related lipid transfer ( start ) domain , which seems to be specific to mammalian evolution .
the n - terminal region of both acots is unrelated to any of the other start - containing enzymes , suggesting that these proteins may be the result of a gene fusion event between an acyl - thioesterase and start domain - containing sequences . like acot8 ,
blast results did identify 22 per cent sequence identity with thioesterase superfamily member 4 ( them4 is the official name ) -- also known as akt c - terminal modulator protein ( ctmp ) .
this small degree of homology suggests that the two proteins may share common evolutionary ties .
another protein , thioesterase superfamily member 5 ( them5 ) shares more than 37 per cent identity with them4 but lacks significant sequence homology with acot13 .
similar to acot13 , both them4 and them5 contain a single ' hot dog ' domain .
furthermore , type ii proteins , them4 and them5 do not share sequence homology with other ' hot dog ' domain superfamily members .
the ' hot dog ' fold - containing proteins possess highly conserved structural elements that are created by highly divergent sequences .
this high degree of divergence makes evolutionary comparisons difficult without three - dimensional structures , because conservation of structural interactions is not directly associated with residue conservation .
a cladogram depicting the evolutionary relationships observed between human , mouse and rat type ii protein is shown in figure 2b .
the mouse genome contains an additional type ii acot , acot10 , which shares approximately 95 per cent mrna nucleotide identity with acot9 .
unlike acot3 and acot5 , however , which are found in both mouse and rat , acot10 has only been identified in mice , indicating that the gene duplication event occurred less than 17 mya -- that is , after the mouse - rat split . in mouse , acot9 is located at chromosomal location x f3 and acot10 is found on 15 a2 .
the amplification and movement of genes to a new chromosomal location is often facilitated by transposable elements , as opposed to duplications occurring within close proximity on a chromosome , which are attributed to errors during meiosis .
the remaining seven type ii acot genes are highly conserved among human , mouse and rat , confirming that these genes were all present in the ancestor prior to the mammalian radiation .
type i and type ii acyl - coa thioesterases represent two structurally distinct enzyme groups .
an unrooted dendrogram -- created using protein sequences from both type i and type ii enzymes , closest - related homologues ( baat , them4 and them5 ) , distant /-hydrolase fold homologues ( ces1 , ache ) and -actin ( actb ) as an unrelated sequence -- exemplifies how dissimilar the two groups are from one another ( figure 3 ) .
it also illustrates how the type ii proteins must have originated from a common , evolutionarily distant , predecessor ( as reflected in individual branch proximity to the origin of the tree ) .
bile acid acetyl co - a : amino acid n - acyltransferase ( baat ) , thioesterase superfamily members 4 and 5 ( them4 and them5 ) are the closest - related homologues to type i ( baat ) and type ii sequences ( them4 and them5 ) .
type i acot branches are highlighted in blue , whereas type ii are in red .
acot1 , acot2 and acot4 contain an n - terminal acyl - coa thioester hydrolase domain ( pfam 04775 ) .
this domain does not participate directly in catalysis , but studies indicate that the n - terminus plays an important role in regulating enzyme activity .
all type i proteins also contain a c - terminus esterase - lipase superfamily domain .
all residues required for catalysis are located within this domain , which is also known as the /-hydrolase fold .
proteins that share this common structural element make up the functionally diverse /-hydrolase fold superfamily of enzymes . within this family
are a number of other distantly related proteins that exhibit esterase activity , including carboxylesterases , ache and lipases .
the superfamily has diverged greatly and many members share very little sequence homology but are considered evolutionarily linked , based on structural homology .
the enzyme activity is dependent on a catalytic triad composed of highly conserved serine , histidine and aspartate residues found within the active site .
the crystalline structure for acot2 indicates that the enzyme is most likely biologically active as a monomer .
the structure also reveals that the n - terminal acyl - coa thioester hydrolase domain is connected to the active site by a long loop ; it is postulated that this contributes substrate specificity , although this has yet to be verified experimentally .
genomic analysis suggests that acot6 should encode a similar 3 exon transcript ; however , the only detectable transcript is truncated and utilises a start codon located within the 3 ' end of exon two , resulting in the synthesis of a truncated protein .
in addition to the four protein - coding genes , there is one intronless pseudogene , referred to as acot4p , located on chromosome 19q13.12 .
acotp4 is most likely the result of retroviral action that has inserted this dna segment elsewhere in the genome .
acot1 is cytosolic and primarily responsible for the metabolism of long - chain acyl - coas .
acot2 and acot1 can metabolise a similar range of substrates ; however , acot2 contains an n - terminal mitochondrial targeting signal which directs its transport to mitochondria .
acot4 contains a c - terminal peroxisomal targeting signal , directing transport to peroxisomes , where it exhibits high activity against succinyl - coa , as well as a wide variety of short - chain dicarboxylic - coas .
as previously mentioned , acot6 lacks the n - terminal acyl - coa thioester hydrolase domain .
although studies have yet to confirm the enzymatic activity of acot6 , the fact that it contains a complete c - terminal esterase - lipase domain supports its role as a functional esterase .
acot6 is predicted to be cytosolic , due to a missense mutation which alters the peroxisomal targeting signal that is found in the acot6 mouse ortholog .
the subcellular localisation and substrate specificity for each enzyme suggest that each plays an important , non - redundant function within the cell .
the type ii enzymes are more distantly related than type ii acots , as indicated by the relatively low degree of sequence conservation observed between proteins . despite the low degree of sequence homology , the enzymes are structurally related and capable of metabolising similar substrates .
acot7 , formerly called brain acyl - coa hydrolase ( bach ) , has been extensively characterised , owing to recent studies that indicate arachidonoyl - coa as a preferred substrate .
the metabolism of arachidonoyl - coa by acot7 gives rise to arachidonic acid and coenzyme a. arachidonic acid serves as a precursor for a number of compounds associated with inflammation , including prostaglandins , leukotrienes and thromboxanes .
acot8 is found in peroxisomes , where it metabolises a diverse range of acyl - coa compounds .
acot8 also has high activity towards branched- and methyl - branched chain acyl - coas .
acot9 contains a mitochondrial - targeting signal and metabolises medium- to long - chain acyl - coas .
acot9 exhibits the highest activity towards myristoyl - coa , which can then be used by n - myristoyltransferase to modify proteins posttranslationally .
expression of mouse acot11 , formerly referred to as brown fat - inducible thioesterase ( bfit ) , increases after cold shock .
this protein was found to play a role in regulating thermogenesis , which helps to maintain body temperature .
the start domain in animals is commonly found within homeodomain transcription factors , where lipid - binding regulates transcription .
the binding of lipids to the start domain in acot11 or acot12 acts as a mechanism for regulating activity and for allowing for rapid enzyme activation .
acot13 , previously called thioesterase superfamily member 2 ( them2 ) , differs from the other type ii acots , in that it contains a single ' hot dog ' domain .
were recently determined using recombinant protein ; results indicate that acot13 prefers medium- to long - chain - acyl - coa thioesters .
this finding suggests that the start domains , identified in acot11 and acot12 , could have a similar effect on enzyme activity .
recent in vitro kinetic activity studies revealed that them4 is a broad - range , high - activity acyl - coa thioesterase .
similar to acot13 , them4 preferentially metabolises medium- to long - chain acyl - coa thioesters .
as such , we recommend that the enzyme be renamed acot14 according to the accepted nomenclature established by hunt et al . .
acyl - coa thioesterase activity has not been verified for them5 ; however , activity is probably based on the high degree of sequence identity with them4 , but the exact role that this protein might play in fatty acid metabolism has yet to be determined .
the human acyl - coa gene family represents a diverse group of enzymes that catalyse the hydrolysis of acyl - coa thioesters to their corresponding free fatty acids and coenzyme a. these enzymes are involved in a number of cellular processes but are primarily thought to play an important role in lipid metabolism . despite catalysing the same enzymatic reaction , the acyl - coa thioesterase family members can be divided into two distinct groups , type i and type ii . the two groups do not share common structural elements or sequence homology and are therefore considered analogous , rather than homologous , enzymes ; this is probably another example of convergent evolution .
type i proteins belong to the /-hydrolase fold superfamily , whereas type ii proteins fall into the ' hot dog ' fold superfamily . to date , four type i and six type ii genes have been identified within the human genome .
there are two additional genes , them4 and them5 , which gene products share both structural and sequence homology with other type ii proteins .
them4 was identified as having acyl - coa thioesterase activity and should be considered as a new family member . in the future , perhaps them5 should also be considered as a type ii acot family member .
recent studies have greatly expanded the current understanding of acyl - coa thioesterases ; however , many precise physiological functions are not completely understood and warrant further investigation .
we would like to thank our colleagues for valuable discussion and critically reviewing this manuscript .
this work was supported , in part , by the following nih grants ; r01ey17963 and r21aa017754 ( v.v . ) and p30es06096 ( d.w.n . ) .
 the acyl - coa thioesterase gene ( acot ) family encodes enzymes that catalyse the hydrolysis of acyl - coa thioester compounds , also known as activated fatty acids , to their corresponding non - esterified ( free ) fatty acid and coenzyme a ( coash ) . 
 these enzymes play a very important role in lipid metabolism by maintaining cellular levels and proper ratios of free and activated fatty acids , as well as coash . within the acyl - coa family 
 there are two distinct subgroups , type i and type ii . despite catalysing the same reaction , 
 the two groups are not structurally similar and do not share sequence homology , strongly suggesting convergent evolution . 
 this suggestion is further supported if one compares the human with the mouse and rat acot gene families . to date , four human type i acots have been identified which belong to the /-hydrolase fold enzyme superfamily . 
 type ii acots fall into the ' hot dog ' fold superfamily . 
 there are currently six human type ii genes ; however , two homologous proteins , thioesterase superfamily members 4 ( them4 ) and 5 ( them5 ) share common type ii structural features and , in the case of them4 , acyl - coa thioesterase activity -- suggesting that the family may be larger than previously realised . 
 although recent studies have greatly expanded the current understanding of these proteins and their physiological importance , there are a number of members whose functions are relatively unexplored and which warrant further investigation . 
Introduction
Human 
Type I ACOT structures
Type II ACOT structures
Conclusions
Acknowledgements