carboxyl/cholinesterases: a case study of the evolution of a successful multigene family

Carboxyl/cholinesterases: acase study of the evolution of asuccessful multigene familyJ.G. Oakeshott,* C. Claudianos, R.J. Russell,and G.C. Robin

Summary

The evolution of organismal diversity among the Metazoa is dependent on theproliferation of genes and diversification of functions in multigene families. Herewe analyse these processes for one highly successful family, the carboxyl/cholinesterases. One key to the expansion of the functional niche of this group ofenzymes is associated with versatile substrate binding and catalytic machinery.Qualitatively new functions can be obtained by substitution of one or a very fewamino acids. This crudely adapted new functionality is then refined rapidly by apulse of change elsewhere in the molecule; in one case about 13% amino aciddivergence occurred in 5–10 million years. Furthermore, we postulate that theversatility of the substrate binding motifs underpins the recruitment of severalfamily members to additional noncatalytic signal transduction functions. BioEs-says 21:1031–1042, 1999. r 1999 John Wiley & Sons, Inc.

It is now widely accepted that all the proteins in extant lifeforms fall into just a few hundred lineages of structurallydefined and ancestrally related superfamilies.(1,2) It followsthat the evolution of diversity at an organismal level dependson the versatility of many superfamilies in evolving newfunctions from finite starting materials. This paper exploreshow this versatility is achieved and exploited by tracing theevolution of the carboxyl/cholinesterase multigene family, abranch of the a/b-hydrolase fold superfamily.

A versatile esteratic capability was in fact required at thevery outset of organismal evolution. Many fundamental as-pects of cellular functioning, including nucleic acid and lipidmetabolism, require esterase activity. Indeed esterasesevolved independently in several superfamilies, most of themusing distinct reaction mechanisms.(2–5) In addition, the diver-sity of biological esters also dictated a rapid diversification ofesterases within each superfamily. In prokaryotes, enzymesfrom the a/b-hydrolase fold superfamily of proteins haveesteratic capabilities ranging through thio, phospho, and

carboxyl esterases, as well as various peptidase, haloge-nase, and other hydrolytic functions.(6)

The evolution of eukaryotes, in particular Metazoa, re-quired an additional suite of esterase functions. Metabolismand transport of lipid nutrients and other xenobiotic estersbecame more complex, as did the role of lipids in membranefunction. Novel neuronal and hormonal processes also devel-oped, many of them involving esters. Although esterases inother superfamilies were also utilised, the a/b-hydrolase foldenzymes in particular proliferated and diversified. This led tothe evolution of several new a/b-hydrolase fold multigenefamilies more or less specific for carboxylester substrates.(4,7,8)

The carboxyl/cholinesterases are one such family. Theiramino acid identities with other a/b-hydrolase fold proteinsare essentially negligible, and even among several of themajor lineages of carboxyl/cholinesterases identities as lowas 19% occur.(8,9)

The a/b-hydrolase foldThe a/b-hydrolase fold is a single-domain structure definedby a mainly parallel b-sheet of 8–10 b-strands that arearranged in a mainly characteristic order and orientation inthe primary sequence (Fig. 1). The b-sheet lies at the core ofthe folded protein structure; it is variously decorated withloops and helices that are interspersed among the b-strandsin the primary sequence. The molecular weights of the

CSIRO Entomology, Canberra ACT 2601, Australia.*Correspondence to: J.G. Oakeshott, CSIRO Entomology, GPO Box1700, Canberra ACT 2601, Australia. E-mail: [email protected]

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BioEssays 21:1031–1042, r 1999 John Wiley & Sons, Inc. BioEssays 21.12 1031

subunits of a/b-hydrolase fold proteins vary between 25 and65 KDa, depending largely on the extent of the decorations.The carboxyl/cholinesterases are among the largest of theseproteins.

Notwithstanding the sequence divergence among thecarboxyl/cholinesterases, the overall conservation of theirtertiary structure appears to be high. Analyses of the tertiarystructures of two family members with only 28% amino acididentity reveal that the positions of about 400 of 540 residuesoverlap within a couple of Ångstroms.(6) Most of the diver-gence in the tertiary structure occurs in the decorations on thesurface of the protein.

Almost all the catalytically active a/b-hydrolase fold en-zymes are believed to use a two-step reaction mechanismbased on a ‘‘catalytic triad’’ of residues that are noncontigu-ous in the primary sequence but adjacent in the tertiarystructure.(6,9) In the carboxyl/cholinesterases, the triad isSer-His-Glu, or, less commonly, Ser-His-Asp. The first step ofthe reaction liberates the alcohol moity of the substrate andforms a covalent linkage between the remaining acid moity ofthe substrate and the Ser. The second step cleaves thislinkage and liberates the acid moity of the substrate, largelythrough the action of the His. Similar catalytic triads are foundin some other superfamilies containing esterases, but theorganisation of the triad residues in the primary sequenceand active site is completely different, so their functionalsimilarity is interpreted as convergent evolution.(6)

The catalytic triad of well-studied carboxyl/cholinesteraseslies at the base of a deep catalytic gorge.(9–11) At least forsome members of the family, the depth of the gorge (about 30Å) has necessitated the evolution of peripheral substratebinding sites at the lip of the gorge and a set of guidanceresidues down the lining of the gorge. The binding ofsubstrate at the lip activates conformational changes thatfacilitate movement of substrate down the gorge. This elabo-rate machinery enables extremely efficient kinetics; the kinet-ics of the reaction of acetylcholinesterase (AChE) with acetyl-choline approaches the theoretical maximum.(12) The identitiesof the binding and guidance residues can vary greatly amongfamily members, however, with profound consequences forthe kinetics of the enzyme. Comparisons of a carboxyl andcholinesterase suggest tenfold differences in the volume ofthe gorge and major differences in its charge.(13) One of ourobjectives in this review is to analyse how the sequence andphysicochemical differences in structures like the catalyticgorge and substrate binding sites generate the great diversityof substrate specificities and reaction kinetics that haveevolved among family members.

Intriguingly several members of the carboxyl/cholinester-ase multigene family have now been reported which lack afunctional catalytic triad(14–17) and thus lack esteratic activity.However, some of these enzymes have ligand-binding func-tions involved in signal transduction. In some cases thesefunctions may be mediated by unrelated domains fused to the

Figure 1. Stereo diagram of the atomic structure of acetylcholinesterase from Torpedo californica,(10) looking down the catalytic gorgeinto the active site. Fourteen aromatic residues lining the gorge are shown in yellow and the catalytic triad is shown in orange (Ser),magenta (Glu), and purple (His). Three substrate binding domains surrounding the gorge on the surface of the molecule are colouredblue. The major b-sheet is coloured green. To aid clarity the aromatic gorge residues and catalytic triad are shown as space-filled, and theperipheral binding sites are shown as stick format, with the remainder of the protein as a ribbon diagram.

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carboxyl/cholinesterase domain, but in others it may be dueto the active site of the carboxyl/cholinesterase domain.

Phylogeny of the carboxyl/cholinesterasesCarboxyl/cholinesterase genes occur sporadically in bacterialgenomes, either on chromosomes or plasmids (Fig. 2). Five

have now been found in diverse cyano-, eu-, and archaebac-teria.(18–21) Eight of the 11 chromosomal genomes frombacteria that have now been fully sequenced lack anycarboxyl/cholinesterases, however.(20) Clearly, the familyevolved in ancient bacteria but has not proliferated in manyextant prokaryotes.

Nor has the family proliferated in lower eukaryotes (Fig. 2).Only one member occurs in the one yeast genome se-quenced completely (Saccharomyces cerevisiae).(22) Thefamily does include some fungal lipases, five from the oneCandida species.(23) Two family members are present in theslime mould, Dictyostelium discoideum.(24)

No carboxyl/cholinesterase gene has yet been isolatedfrom a plant although they obviously existed in antecedentlineages and some other a/b-hydrolases with esterase activ-ity have been identified.(25) Such genes may yet emerge in theArabidopsis thaliana genome project (11% complete at thetime of writing), but their absence from plant databases todate suggests that they have not proliferated in the plantkingdom.

The great majority of known carboxyl/cholinesterase genesare from higher eukaryotes (Fig. 2). Fifty-seven have beenfound in the nematode, Caenorhabditis elegans (85% of thegenome sequenced at the time of writing). A further three notyet in that database have been obtained from directed cloningefforts.(26) C. elegans is among the smallest of higher eukary-otic genomes.(27) Genome projects for other eukaryotes areless advanced, but directed cloning efforts have alreadyidentified about 25 paralogous family members in insects andover 40 in mammals.(28,29)

The distribution of this gene family across organisms isexplained most simply by assuming that at least a fewlineages of carboxyl/cholinesterases differentiated before theprokaryote/eukaryote split, around 2000 Mya.(30) Otherwiseall the prokaryote representatives must be ascribed to morerecent horizontal gene transfer events. Although the latterpossibility cannot be discounted completely, the former issupported by a reconstruction of the phylogeny of 140paralogous carboxyl/cholinesterases for which sequence dataare available (Fig. 2). Note, in particular, that three of the mostdistinct lineages in the phylogeny (20–22% amino acididentities) are constituted from representatives of three majordivisions of the Prokaryota. Certainly several major lineagesin the family must have arisen before or during the earlystages of metazoan differentiation, around 750 Mya,(30) be-cause they each contain nematode, insect, and/or vertebraterepresentatives.

It is also clear, however, that some major radiations haveoccurred subsequent to the separation of the major classes ofMetazoa. Two large lineages in Figure 2 contain 33 C.elegans proteins, but no representatives from other taxa.Over half of about 25 insect proteins ascribed to the family lie

Figure 2. Phylogeny of the carboxyl/cholinesterase multi-gene family. Most of the sequences for the 140 proteinsanalysed can be found in the Pfam,(70) C. elegans (http://www.sanger.ac.uk/Projects/C_elegans/blast_server.shtml) andCOG(20) NCBI databases. Key references are given in the textand the full data set is available from the authors on request.Sequences were aligned using the Pileup program of theGenetics Computer Group (GCG),(71) with default settings(gap weight 3.0 and gap length weight 0.1). A distanceneighbour joining tree was then created using PAUP*(72) inorder to obtain confidence values (*) which have greater than70% resampling frequency from 100 bootstrap replications.Lineages are colour coded to show broad taxonomic group-ings. Terminal lineages containing multiple paralogous se-quences are indicated by (d). A full presentation of thephylogeny for 49 sequences in the C. elegans database isgiven in Figure 3B. (Eight sequences in that database thathave not yet been localised to chromosomal contigs areexcluded from this and Figure 3). Note that the C. elegansAChE lineage contains AChE-1, which is in the C. elegansdatabase (and denoted gene 27 in Figure 3) and AChE-2,AChE-X and AChE-Y, which have not yet been recovered fromthe genome project.(26) CE 5 carboxylesterase. The verte-brate CES1–CES4 groups are those of Satoh and Ho-sokawa.(29)

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in one lineage, with most of the remainder having relativelyold and/or specialist functions like AChE and the noncatalyticglutactin and neurotactin. Almost all of 40 vertebrate mem-bers of the family lie in just two tightly contained segments ofthe phylogeny, again excepting some relatively old and/orspecialist members such as the cholinesterases, thyroglobu-lin, and a deacetylase.

Specialist proteins in some lineages within the phylogeny,such as the cholinesterases, may have retained very similarfunctions, and over 40% sequence identity, for several hun-dred million years of organismal evolution. Comparativestudies, however, show that other lineages have spawnednew members that postdate the origins of families, genera oreven modern-day species.(28,31,32) Some of the well-studiedinsect sublineages contain paralogues that separated in thelast 50 Myr but now have as little as 60% sequence iden-tity.(28,33,34) One key feature underlying the ongoing diversifica-tion of this multigene family is thus a capacity to changesequence rapidly and expand into new biochemical nicheswithin the same timeframe that their host organisms developsnovel ecological niches.

Another feature of the phylogeny is that the most similarsequences within an organism are generally physically co-located on its chromosomes. This is best demonstrated in C.elegans where 29 of the 49 carboxyl/cholinesterase genesthat have been mapped to chromosomal contigs are locatedat just six distinct chromosomal sites (Fig. 3). Almost withoutexception, these gene clusters correspond to individual radia-tions in the phylogeny. Clearly, the processes that increasethe copy number of the multigene family generally involvephysically local amplifications. We now analyse in more detailhow two clusters have developed.

The a-cluster in higher DipteraIn D. melanogaster the a-cluster comprises ten active ester-ase genes plus one pseudogene (Fig. 4)(35,36) that arescattered irregularly over 60 kb and interspersed with at leasttwo unrelated genes. The amino acid sequences of theesterases encoded by the cluster have diverged significantlyfrom one another (37–66% amino acid identities), but theyalso share a distinctive combination of features. Thesefeatures include several small insertions/deletions and aunique 12 residue motif in a presumptive peripheral substratebinding site. In most cases there is no secretion signal but inseveral there is an endoplasmic reticulum retention signal.Although not contiguous in the primary sequence, the resi-dues lining the gorge are also characteristic. Additionally, thea-cluster genes possess a unique complement of introns.

Two esterase isozymes encoded by the cluster have beencharacterised biochemically.(7,37) They are termed EST9 andEST23. Both are monomeric proteins of 60–65 kDa withbroad expression profiles across tissues and life stages. Invitro substrates include esters of carboxylic acids with small

aliphatic or aromatic alcohols. In vivo functions are unknown,but there is evidence from orthologues in other species thatloss of EST23 activity reduces fitness.(38) Significantly, thetwo enzymes are distinct biochemically: EST9 is cytosolicand the other, EST23, is membrane bound. The mobility ofEST23 is about 30% that of EST9 under native polyacryl-amide gel electrophoresis (PAGE). EST9 is sensitive toinhibition by carbamates and mercuric compounds, whereasEST23 is not. EST9 is also orders of magnitude moresensitive than EST23 to inhibition by organophosphates(OPs). Indeed, on the inhibitor based classification of Healy etal,(39) EST23 is a subclass I carboxylesterase and EST9 asubclass II cholinesterase. Thus, the sequence differencesamong cluster members are associated with qualitative bio-chemical differences.

Parts, or all, of the a-cluster have also been isolated fromD. buzzatii, which is in a different Drosophila subgenus fromD. melanogaster, plus Lucilia cuprina and Musca domestica,which are in different families within the higher Diptera (Fig.4).(28,33,34,40) Orthologues for several of the D. melanogastergenes are present, and conserved in order and orientation,across the other three species. This is not true, however, forat least three genes in this cluster. aE1 differs in position and

Figure 3. A: Chromosomal arrangement of 49 carboxyl/cholinester-ase genes localised to chromosomal contigs in the C. elegansgenome project at the Sanger Centre (http://www.sanger.ac.uk/Projects/C_elegans/webace_front_end.shtml). Genes were identi-fied by homology based searches of the C. elegans DNA sequenceand Wormpep databases (see Figure 2) using the lipase fromGeotrichum candidum,(73) aE9 from Drosophila melanogaster(36) andAChE from Torpedo californica.(12) Note that the cloned Ace-2(chromosome I), Ace-x and Ace-y (close together on chromosome II)sequences of Arpagaus et al.(26) are not included because they havenot yet been recovered from the genome project and cannottherefore be assigned precise locations. Also excluded are eightother carboxyl/cholinesterase sequences that have been identifiedbut not yet localised to a contig in the genome project. Alpha-numericcodes indicate the cosmid designation of the localised genes in thedatabase. Where the lines cross the chromosome the genes arecontiguous but on opposite DNA strands. Chromosome number isindicated in Roman numerals, and the scale of the chromosomalcontigs (in Mb) is rooted at the centromeres. Some groups of genesare shown preceded by putative trans-spliced leader sites (♦)(74)

which direct transcription of polycistronic message. Colour codingindicates four sets of phylogenetic lineages in Figure 3B. B: Phyloge-netic relationships of C. elegans carboxyl/cholinesterases in Figure3A. The tree indicates four sets of lineages, two of which (red andblue) show extensive gene duplication events in Figure 3A. Theproteins R173.3, W09B12.1, and F13H6.3 correspond to ges-1,(75)

Ace-1,(26) and Est2,(76) respectively. T28C12.4A and T28C12.4B areproducts of a single alternatively spliced gene at the T28C12.4 site inthe database and C23G4.3 and C23H4.3.1 are the products of twopreviously unresolved genes at the C23H4.3 site. Gene numbersnext to the full alpha-numeric nomenclature are for cross-referencingto Figure 2.

=

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orientation between the drosophilids; there are at least fourcopies of aE1 and/or aE2 in M. domestica; and aE4 hasduplicated independently in D. melanogaster and D. buzzatii.Notably, the most recent copy of aE4 in D. melanogaster wasoriginally inserted intact into an intron of aE6, but hassubsequently been inactivated. Thus there have been severalqualitative changes in the composition of the cluster in the80–100 Myr of organismal evolution encompassed by thefour species of flies.

A robust phylogeny of the a-cluster genes can be con-structed from the sequence data for the four species andpresence/absence data on several indels and five in-trons.(33,34) This shows that the cluster has been assembledby a series of single gene duplication events. These eventsgenerally, but not always, deposit the new copy of the gene atan adjacent locality and similar orientation within the cluster.

Further work on the most recent copy of the aE4 gene inthe D. melanogaster lineage(33) also informs of the processesof gene loss within the cluster. The gene is present in anactive form in D. yakuba and D. simulans, two close relativesof D. melanogaster. Thus inactivation in D. melanogaster isrelatively recent, and must have occurred within the 2.5–5Myr that separate this species from D. simulans.(41) Neverthe-less, in this time this copy of aE4 has accumulated fiveindependent inactivating mutations and over 20 other nonsyn-onymous mutations. This nonsynonymous rate of change is

as high as, if not higher than, the presumptively neutral rate ofsynonymous change. Most of the inactivating mutations aredeletions, and it is estimated that less than half the DNA in thegene would survive for more than 10 Myr. This agreesremarkably well with estimates of about 12 Myr from analysesof inactivated transposable elements in Drosophila, albeitthat some estimates from vertebrate genes are several-foldlarger.(42,43) Clearly, genes can be lost without trace quitequickly during cluster evolution, at least in Drosophila.

Comparisons of the D. melanogaster and D. buzzatii datasets reveal several-fold differences in rates of sequencechange across paralogous cluster members.(28,33,34) Interest-ingly, the slowest evolving gene, aE9, also has the mostdistinctive expression profile in the cluster. The activity of aE9is limited largely to embryos, whereas other genes from thea-cluster have predominantly larval and adult pulses ofactivity. At the other extreme, orthologous amino acid identi-ties for five members of the cluster are only about 60 and75%. As the two species separated about 50 Mya,(41) thisrepresents a higher rate of change than that for most singlecopy (nuclear) genes.(7,17) Perhaps fast rates of change areexpedited in gene clusters because of the relaxation ofconstraints on individual genes due to the back-up providedby others. Consistent with this, some of the fastest evolvinga-cluster genes are aE1, aE2, and aE4, the same genes fromwhich duplicate copies have arisen within the higher Diptera.

Figure 4. Genes of the a-cluster identified to date from Drosophila melanogaster, Drosophila buzzatii, Musca domestica, and Luciliacuprina. The whole of the cluster has been recovered from D. melanogaster and probably also D. buzzatii, but it is only partlycharacterised in M. domestica and in particular L. cuprina. a-Esterase genes not known to vary in position or orientation across the fourspecies are shown as black arrows; those known to vary in position or orientation are shaded grey. Non-esterase genes are unshaded(ubc 5 ubiquitin conjugating enzyme, trop 5 putative tropomyosin). The contig in D. melanogaster is about 60 kb. Line breaks indicateone uncharacterised gap in the D. buzzatii data and five in M. domestica, while no contig has yet been established for L. cuprina. Theorientations of some genes are therefore unknown and accordingly shown as blocks rather than arrows. There is some ambiguity aboutthe orthologues of the Drosophila aE1 and aE2 genes in the other two species so the candidates are shown as aE1/2.

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The Drosophila b-clusterThe b-cluster is best characterised in D. melanogaster, whereit comprises just two genes, Est6 and Est7 (Fig. 5).(44,45) Theircoding regions are similarly oriented and only 197 bp apart.Furthermore, the coding region of the upstream Est6 genecontains several of the promoter elements for the down-stream Est7 gene. The EST6 and EST7 proteins are bothsoluble, secreted, and glycosylated monomers of about 60kDa that are detected after native PAGE using standardnaphthyl acetate substrates. EST6 and EST7 have onlyabout 40% amino acid sequence identity, however, and theirexpression patterns are quite distinct. EST6 is expressedprincipally in the haemolymph of larvae and adults and in thesperm ejaculatory duct of adult males,(39,46) whereas EST7 islocalised to cuticular tissue of late larvae or early pupae.(45)

The functions of EST7 and EST6 in the haemolymph areunknown. EST6 expressed in the ejaculatory duct, however,is transferred to the female during mating, where it acts withother seminal fluid components to trigger egg laying andrepress remating behaviour.(17)

Est6 and to a lesser extent Est7 have also been character-ised in other members of the melanogaster group of species,and in D. pseudoobscura, which is in a different speciesgroup (obscura), albeit the same subgenus (Sophophora)

(Fig. 5).(17,32,46) At least one copy of each gene is present in allthe species studied. However, monomeric quaternary struc-ture and high expression of EST6 in the ejaculatory duct havebeen acquired recently, specifically, within the three siblingspecies in the melanogaster subgroup. Also, D. pseudoob-scura has two adjacent and similarly oriented copies of Est6.There is evidence of a conversion event involving parts of thetwo copies, and their nonconverted parts still show 81%amino acid identity. This suggests a relatively recent origin,probably within the 35 Myr since the differentiation of theobscura species group.(41) The function of the upstream copyis unknown; the downstream copy shows the presumptivelyancestral haemolymph expression associated with Est6 inother species.

Putative b-cluster genes have also been identified from D.buzzatii and D. virilis, which are in the repleta and virilisspecies groups, respectively, of the subgenus Drosophila(Fig. 5).(17,47,48) Classical genetic studies show that bothspecies have very closely linked loci encoding esteraseisozymes with the typical haemolymph and late larval/earlypupal cuticle expression profiles of EST6 and EST7. In D.virilis, the gene for a third isozyme specific for the spermejaculatory bulb (cf the ejaculatory duct for EST6 in D.

Figure 5. Organisation of b-esterase genes in various Drosophila species as inferred from molecular and classical genetics (arrowsand blocks, respectively). Est6 and its putative orthologues are shown in black, Est7 and putative orthologues are unshaded. For themolecular analyses, the arrows indicate direction of transcription. Major expression phenotypes are indicated below the arrows. Thequestion mark indicates unknown linkage and orientation (between the arrows) or unknown expression (below the arrows). ..//.. indicatesclose linkage but precise location unknown. haem 5 haemolymph; ej duct 5 ejaculatory duct; ej. bulb 5 ejaculatory bulb. Adapted fromOakeshott et al.(17)

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melanogaster) maps to the same region. Genomic clones of15–16 kb that contain parts of this cluster have been isolatedfrom both species. In each case, the cloned region containsthree or four separate subregions with homology to Est6 andEst7 and hybridises in situ to the region of the chromosome towhich the isozyme genes have been mapped. Transcriptionalanalysis of one of the D. virilis genes shows it is highlyexpressed in the sperm ejaculatory bulb.

Full sequences have been determined for two of the D.buzzatii genes and the D. virilis Est6-like gene that isexpressed in the ejaculatory bulb.(17,47,48) All three sequencesencode proteins with homology (45–50% identity) to EST6and EST7 throughout their sequences. However, all threealso have at least two inactivating substitutions in the catalytictriad. It seems likely therefore that the b-cluster contains atleast four genes in each species, that at least two of thegenes in D. buzzatii and one in D. virilis produce esteraticallyinactive proteins, and that at least one inactive and one activeprotein in D. virilis are specific for the sperm ejaculatory bulb.Thus, as with the a-cluster, several qualitative changes haveoccurred in the composition and function of the b-cluster inthe 50 Myr since the two subgenera of Drosophila diverged.

Two studies have analysed interspecific sequence varia-tion in the b-cluster in relation to inferred tertiary structures(as modelled on AChE). The first, relatively coarse, analysisinvolved comparisons of EST6 in D. melanogaster with theesteratically active, haemolymph-expressed orthologue in D.pseudoobscura and the esteratically inactive homologuesEST1 and EST5 in D. buzzatii and D. virilis.(17) The residues ineach protein were partitioned into eight structurally defined

categories, and all three comparisons revealed widespreadchange across all categories. The comparison between D.melanogaster and D. pseudoobscura showed no statisticallysignificant differences in the rates of change across catego-ries, although changes in b-sheets and other internal struc-tures were physicochemically more conservative than thoseelsewhere. The other two comparisons revealed three- tofourfold differences in substitution rates across categories,with the internal b-sheets and salt and cysteine bridges mostconserved, and the a-helices and many other loop regionsless so. Residues forming the glycosylation sites, the activesite gorge, and the peripheral binding site were least con-served. The high levels of variation in the latter two categoriesare consistent with the loss of esteratic function in EST1 andEST5.

The second analysis of this type focussed on EST6differences within the melanogaster species group (Fig. 6).(49)

This showed that the internal lineage of the species’ phylog-eny where the monomeric structure and ejaculatory ductexpression were acquired showed more than a twofoldacceleration in the rate of sequence change. Thirteen percentof the protein changed in a period of 5–10 Myr, and anunusually high proportion of the changes were nonconserva-tive physicochemically. Variation in a-helices, glycosylationsites, and two particular b-strands all contributed dispropor-tionately to the accelerated rate of change. The changes inglycosylation can be related to the function of glycans instabilising the ejaculatory duct enzyme on its transfer to thefemales,(50) while some of the other secondary structurechanges may involve the transition from dimer to monomer.

Figure 6. Phylogeny of part of the melanogaster species group of Drosophila with branch lengths proportional to silent site divergencein Est6 and numbers of amino acid replacements in EST6 boxed for each branch. The functional change in EST6 occurred in the internalbranch of the phylogeny between the lineages giving rise to D. yakuba and D. melanogaster. Modified from van Papenrecht.(49)

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Variation in the active site gorge and peripheral substratebinding site did not contribute disproportionately to the accel-erated change, but there is also no direct evidence that thecatalytic function altered.

Molecular bases of functional changesWe see from the above that qualitative shifts in the functionsof particular carboxyl/cholinesterases are associated withwidespread changes in their sequences over short periods ofevolutionary time. Clearly, not all the sequence changes areequally important in effecting the functional shifts. In thissection, we identify specific sequence changes that areprimarily responsible for some functional changes.

Carboxyl/cholinesterase function in several insect speciesis currently shifting in response to the widespread use oforganophosphate (OP) insecticides. In some cases, includingcertain aphids and culicine mosquitoes, resistance to OPshas been achieved by tandem amplifications of particularcaboxylesterase genes to up to 200 or more copies.(31,51,52)

This results in commensurate overexpression of the proteins.Although the protein has negligible OP hydrolytic activity, itsabundance (up to 3% of total protein) enables it to sequestersufficient OP for the organism to survive. More frequently,however, resistance appears to be due to structural mutationsin the proteins rather than changes in their copy number.

One set of structural changes affects the primary target forthe OPs, namely AChE. In the case of wild-type AChE, OPsbind covalently, and essentially irreversibly, with the Ser of thecatalytic triad. Several species have evolved mutant forms ofAChE in which this Ser is immune to inhibition by OPs.(31,53)

The mutations best characterised to date, in D. melanogasterand M. domestica, occur at six sites lining the active sitegorge.(53–55) Two of the substitutions are found in bothspecies, and two are specific to each. Multiple alleles arefound in each species containing different subsets of thesubstitutions, and, in general terms, resistance to OP inhibi-tion increases with the number of substitutions. Molecularmodelling suggests that the mutations constrict the gorge sothat substrate but not OP retains access to the catalytic Ser.

A second set of structural mutations effecting OP resis-tance involves the aE7 gene/enzyme system in the a-clusterof carboxylesterases.(28,34,56–57) In this case, metabolic resis-tance is acquired because the aE7 enzyme acquires theability to hydrolyse OPs. Two resistance alleles have beenwell characterised. These are termed diazinon-type andmalathion-type, but both enable detoxication of broad, over-lapping ranges of oxon OPs. In addition, the malathion-typeconfers especially high resistance to a small number of OPsthat have carboxylester bonds in addition to the phosphoesterlinkages that are characteristic of all OPs. The molecularbases of both alleles have been described for M. domesticaand L. cuprina; essentially the same amino acid changes giverise to resistance in the two species. The OP-susceptible aE7

enzyme is unusually sensitive to inhibition by formation of acovalent bond between the catalytic Ser and the OP. Fordiazinon resistance, the causal change is a Gly = Aspsubstitution in the oxyanion hole of the active site, which isproposed to activate a water molecule needed to hydrolysethe phospho-Ser linkage. The malathion resistant enzymealso has a single residue change, in this case a Trp = Leu orTrp = Ser substitution in the acyl pocket of the active site.This enables limited hydrolysis of the phospho-Ser bondproduced if the phosphate moity in malathion binds the Serresidue, plus improved hydrolysis of the carboxyl-Ser bondproduced if the carboxylester moity of the malathion binds theSer residue.

One important aspect of the AChE and aE7 mutants thatconfer OP resistance is that they represent essentially qualita-tive changes in function. The acquired functions of the newenzymes still leave substantial room for further improve-ments, however. For example, with OP substrates, the ratiokcat /Km (a measure of the adaptedness of the enzyme for thesubstrate) for the diazinon resistant aE7 enzyme is about sixorders of magnitude less than the theoretical, diffusion-limitedmaximum.(28) Given that OP insecticides have only beenwidely used in the last 40 years, the question arises as towhat subsequent mutations would accrue to improve thenewly acquired functions.

There is, in fact, experimental data, albeit for a differentsystem, on the nature of the downstream mutations that effectquantitative improvements in an existing activity of a carboxyl/cholinesterase.(19,58) The system involves the p-nitrobenzylesterase of Bacillus subtilis and the enhanced activity in-volves a carboxylester precursor in the synthesis of anantibiotic. Variation was generated through six generations ofdirected in vitro evolution. This protocol generates mutationsat high frequencies by error-prone PCR, then recombinesmutations by ‘‘DNA shuffling’’ of random fragments. Sixty-foldenhancement of activity was obtained, by the combinedeffects of six amino acid substitutions. Modelling against theAChE structure showed that none of the mutations lie in theactive site, or within 20 Å of the catalytic Ser. Five lie in theperipheral substrate binding site, albeit none appear to beclose enough to interact directly with the substrate. The sixthlies deep within the interior of the enzyme. Thus, improve-ment of an existing activity mainly involves mutation ofresidues in the general proximity of the catalytic machinery,but they need not interact directly with substrate.

The b-cluster of Drosophila is also informative in relationto the molecular basis of functional change. There are at leasta dozen polymorphic residues in EST6 in natural populationsof D. melanogaster.(59) Some of these may be neutral in termsof natural selection, but the frequencies of two variants varysystematically with the latitude of the populations, suggestingthat these two at least are not. These variants differ in

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substrate specificity in vitro and modelling on AChE suggeststhat they lie in the peripheral substrate binding site.(7,17)

The b-cluster also has two other types of polymorphicvariation, which give very different insights into functionalevolution. One of these is a major regulatory polymorphismfor EST6.(60,61) One variant expresses about threefold moreEST6 protein in the sperm ejaculatory duct than the other.Laboratory studies of egg-laying and remating behavioursuggest that this difference affects reproductive fitness. Asmight be expected, the difference is due to polymorphism inthe Est6 promoter but, intriguingly, it is associated with notone but 13 nucleotide differences distributed along a 384 bpsegment of the promoter. This bears out long held suspicionsthat the highly interactive functions of promoter elementscould lead to epistatic selection pressures on many mutationsand, in turn, to the evolution of co-adapted complexes ofpromoter polymorphisms.(62)

The other informative aspect of b-cluster polymorphismsis the existence of several inactivating mutations in the codingregion of Est7 in some lines of D. melanogaster.(43,63) This hasled to the proposition that Est7 is functionless in this lineageand is in the process of becoming a pseudogene.(63) Giventhe proximity of the Est6 and Est7 coding regions and theoverlap in their regulatory elements, we speculate that theadaptive value of the recently acquired ejaculatory ductexpression of Est6 in this lineage may outweigh the functionalsignificance of Est7 and that the latter is now being jettisonedto allow optimisation of the former.

ConclusionsWe have seen that the carboxyl/cholinesterase multigenefamily has expanded to occupy a wide range of functionalniches in the Metazoa. We suggest that this evolutionarysuccess reflects the conservation of two critical structures,which in turn provide the platform for rapid and variedevolutionary responses in others.

One conserved structure is the a/b-hydrolase fold. Conser-vation of this structure appears to underpin the retention of atleast 19% amino acid identity in even the most distantlyrelated family members. Cygler et al.(9) in fact showed that ahigh proportion of the 73 most conserved residues in thecarboxyl/cholinesterases are important to the integrity of theira/b-hydrolase fold. The second highly conserved feature ofthe multigene family is the catalytic triad. This versatilereaction mechanism is used in several superfamilies withunrelated tertiary structures, and it hydrolyses a variety ofbonds even within the a/b-hydrolases. Within the carboxyl/cholinesterases, its predominant use is to cleave carboxyl-ester bonds in a variety of substrate structures, and, more-over, it can be readily redeployed to hydrolyse other linkages.

What then of the nonconserved structures decorating thea/b-hydrolase fold that give effect to changes in substratespecificity? Two key structures in many changes are the

catalytic gorge and peripheral substrate-binding site. Qualita-tive shifts in substrate specificity can be achieved by singleamino acid substitutions in these structures, and quantitativemodifications to the kinetics for the new substrates can thenbe achieved with just a few further substitutions in the samestructures. The outcome may be kinetics that are specialisedfor extreme rapidity, as in AChE, and/or extreme sensitivity,as in many hormone and pheromone esterases.(64,65) Equallythe outcome can be versatility, as in many xenobiotic ester-ases.(29) A key feature of the outcomes in the specialistmembers like AChE and lipases at least is allosteric cooperat-ivity, with substrate binding at the peripheral site causingchanges to the structure and performance of the catalyticgorge. We suspect that the ability of the two structures toachieve a diversity of ligand interactions with relatively fewchanges may also explain why some lineages of the familyhave been co-opted to noncatalytic signal transduction func-tions as well.

One consequence of the ability of the carboxylesterases toacquire at least crudely adapted new functions with relativelyfew changes is that the rate of sequence change is unevenover time. Following the first few changes in critical struc-tures, there is a brief period in which accelerated changespreads more widely through the molecule to refine itsadaptation to the new functional niche. This can result inamino acid sequence divergence of several percent in only afew million years, before the rate returns to a background,‘‘clock-like’’ rate dominated by functionally neutral or nearlyneutral substitutions(17,30) (with obvious parallels to theories ofpunctuated organismal evolution(64)). The process also repre-sents a dramatic demonstration of the covarion (concurrentlyvariable codon) hypothesis, which proposes that the probabil-ity of variation in particular residues of a protein is constrainedby the variation in residues with which it interacts.(67,68)

In terms of genetic mechanisms, data from the carboxyl/cholinesterases confirm widely accepted views that func-tional evolution is associated with gene duplication events.(69)

Comparative analyses of invertebrate gene clusters showthat changes in their composition occur rapidly and recur-rently. Genes within the a-cluster that are diverging mostquickly at the sequence level are also the ones most ofteninvolved in duplication events. However, functionally redun-dant genes in the invertebrate clusters appear to be rapidlyinactivated and lost from the genome. It would seem thatfunctionally redundant templates may only be transientlyavailable for the evolution of new functions. Indeed, evidencefrom some invertebrates indicates that some recently ac-quired functions have been superimposed on existing func-tions, with duplication events yet to take place.

Finally, we note that the evolution of a new enzymefunction will often require a qualitative shift in its regulation aswell as its structure. All the data available on promotermechanics shows the importance of interactions among

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co-adapted promoter elements and it is suspected that thiswill constrain the evolution of new regulatory phenotypes.(62)

The Est6 polymorphism bears this out; two major regulatoryphenotypes in D. melanogaster are associated with a suite ofnucleotide differences in the promoter. Empirical data onregulatory evolution are still few, and it is currently unclearhow a suite of co-adapted promoter differences evolves, andhow it ultimately constrains the evolution of new proteinfunctions. In general terms, however, we suggest that theprinciples of the covarion hypothesis(67,68) are as applicableamong promoter elements and, in some cases, betweenpromoter and coding sequences, as they are within codingsequences.

AcknowledgmentsWe thank Karen Bell, Bronwyn Campbell, Chris Coppin,Narelle Dryden, Rebecca Harcourt, Richard Newcomb, andWendy Odgers for all their various inputs into this review.

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carboxyl/cholinesterases: a case study of the evolution of a successful multigene family

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