Marion J. Healy

Evolutionary genetics of Drosophila esterases

Over 30 carboxylester hydrolases have been identified inD. melanogaster. Most are classified as acetyl, carboxyl or cholinesterases. Sequence similarities among most of the carboxyl and all the cholinesterases so far characterised fromD. melanogaster and other eukaryotes justify recognition of a carboxyl/cholinesterase multigene family. This family shows minimal sequence similarities with other esterases but crystallographic data for a few non-drosophilid enzymes show that the family shares a distinctive overall structure with some other carboxyl and aryl esterases, so they are all put in one superfamily of /β hydrolases. Fifteen esterase genes have been mapped inD. melanogaster and twelve are clustered at two chromosomal sites. The constitution of each cluster varies acrossDrosophila species but two carboxyl esterases in one cluster are sufficiently conserved that their homologues can be identified among enzymes conferring insecticide resistance in other Diptera. Sequence differences between two other esterases, the EST6 carboxyl esterase and acetylcholinesterase, have been interpreted against the consensus super-secondary structure for the carboxyl/cholinesterase multigene family; their sequence differences are widely dispersed across the structure and include substantial divergence in substrate binding sites and the active site gorge. This also applies when EST6 is compared across species where differences in its expression indicate a difference in function. However, comparisons within and among species where EST6 expression is conserved show that many aspects of the predicted super-secondary structure are tightly conserved. Two notable exceptions are a pair of polymorphisms in the substrate binding site of the enzyme inD. melanogaster. These polymorphisms are associated with differences in substrate interactionsvitro} and demographic data indicate that the alternative forms are not selectively equivalentin vivo.

Biochemical and physiological studies of soluble esterases fromDrosophila melanogaster

Twenty-two soluble esterases have been identified inD. melanogaster by combining the techniques of native polyacrylamide gel electrophoresis and isoelectric focusing. The sensitivity of each isozyme to three types of inhibitors (organophosphates, eserine sulfate, and sulfydryl reagents) identified 10 as carboxylesterases, 6 as cholinesterases, and 3 as acetylesterases. Three isozymes could not be classified and no arylesterases were identified. The carboxyl- and cholinesterases could each be further divided into two subclasses on the basis of inhibition by organophosphates and sulfhydryl reagents, respectively. Cholineand acetylesterases have characteristic substrate preferences but both subclasses of carboxylesterases are heterogeneous in substrate utilization. Subclass 2 carboxylesterases exhibit diverse temporal expression patterns, with subclass 1 carboxylesterases generally found in larvae and subclass 1 cholinesterases and acetylesterases more characteristic of pupae and adults. Tissues showing the greatest number of isozymes are larval body wall (eight) and digestive tract (six in larvae, six in adults). Carboxylesterases are distributed across a wide range of tissues, but subclass 1 cholinesterases are generally associated with neural or neurosecretory tissues and subclass 2 cholinesterases with digestive tissues.

Characterisation of juvenile hormone esterase in Drosophila melanogaster

Two approaches were taken to identify and characterise the juvenile hormone esterase (JHE) of Drosophila melanogaster. The first investigated the properties of an esterase denoted EST20, which can be visualised on a native polyacrylamide gel using an in vitro esterase, naphthylacetate. D. melanogaster EST20 is likely to be a homologue of the p-esterase of D. virilis which has been claimed to be JHE. However, we show here that juvenile hormone (JH) hydrolytic activity is not associated with either EST20 in D. melanogaster or the p-esterase in D. virilis. The second approach used a direct radiometric assay of JH-hydrolysing activity. Hydrolytic activity measured in this way was recovered after native PAGE of D. melanogaster pupae did not correspond to any of the naphthylacetate staining esterases. The developmental profile of JH-hydrolytic activity in D. melanogaster correlates inversely with JH titre and closely resembles the lepidopteran profile. Two JH-hydrolysing enzymes were identified; JHE and juvenile hormone epoxide hydrolase (JHEH). The largest peak of JHE activity is soluble and coincides with pupation. Lower JHE activity is found in adult microsomes, together with JHEH activity. JH-hydrolytic activity in larvae is largely due to JHEH. JHEs from D. melanogaster and D. virilis are sensitive to inhibition by the esterase inhibitor OTFP, and, in contrast to lepidopterans, are also sensitive to DFP.

PURIFICATION AND KINETIC CHARACTERISATION OF JUVENILE HORMONE ESTERASE FROM DROSOPHILA MELANOGASTER

Juvenile hormone esterase (JHE) from the prepupal stage of Drosophila melanogaster was purified about 429-fold to near homogeneity by selective precipitations, isoelectric focussing, anion exchange and gel filtration chromatography. The KM and Vmax of the purified enzyme for juvenile hormone III (JHIII) hydrolysis are 89 nM and at least 590 nmol/min/mg, respectively. JHE also hydrolyses the artificial substrate alpha-naphthyl acetate with a KM of 120 micro M and a Vmax of at least 70 mumol/min/mg. Competition of JHIII hydrolysis by five juvenile hormones and twenty-four JH analogues showed JHE is highly selective for JHIII and JHIII bisepoxide (JHP3), and both may be in vivo substrates. Binding in the active site of JHE is promoted by structural features found in JHIII and JHB3 including the epoxide groups in their natural orientations, methyl (rather than ethyl) side-chains, and the 2E, 3 double bond that is conjugated with the ester group. Binding is reduced by almost any departure from these structural features of JH. Co-incubation of the haemolymph JH binding protein, lipophorin, with JHE indicates lipophorin might modulate JH hydrolysis by competition for binding of JH.

CHARACTERIZATION OF THE ESTP PROTEIN IN DROSOPHILA MELANOGASTER AND ITS CONSERVATION IN DROSOPHILIDS

The β-esterase cluster of D. melanogaster comprises two tandemly duplicated genes. Est6 encodes the well-characterized 5′ gene, but the product of the second gene, denoted EstP, had not previously been identified. Here we show that the EstP gene encodes the carboxylesterase EST7. Expression of EstP using the Baculovirus system led to production of a carboxylesterase biochemically indistinguishable from EST7. Furthermore, a naturally occurring EstP variant produces greatly reduced amounts of EstP mRNA and no detectable EST7 protein. Finally, introduction of a wild-type copy of EstP by germline transformation into the variant strain confers the wild-type EST7 phenotype. We show that EST7 differs from EST6 in its substrate and inhibitor specificities and tissue distribution. Germline transformation experiments show that EstP expression is controlled by sequences located between 192 bp 5′ and 609 bp 3′ of the EstP coding region. Data comparisons with other drosophilid esterases suggest that the site of expression, and hence the function, of EST7 has been conserved across lineages in both the subgenera Drosophila and Sophophora.

Molecular insights into the evolution of an enzyme; esterase6 in Drosophila

It is still a suspicion among some evolutionary biologists that the incursion of molecular biology into their field will do little more than determine the DNA sequence differences underlying evolutionary changes already evident at the organismal level. Work on an esterase enzyme involved in the reproductive biology of Drosophila belies this view. Although it is already one of the most intensively studied gene - enzyme systems at an organismal level, recent molecular invetigations reveal several unexpected, and, in some cases, still inexplicable phenomena in its evolutionary history.

Associations of esterase 6 allozyme and activity variation with reproductive fitness inDrosophila melanogaster

Previous studies have shown that the esterase 6 (EST6) enzyme ofD. melanogaster is mainly produced in the sperm ejaculatory duct of the adult male and comparisons of wild-type males with laboratory null mutants have suggested that the enzyme plays a role in reproductive fitness. In this study we have compared 18 field-derived lines each isoallelic forEst6 for differences in five components of male reproductive fitness. No consistent fitness differences were found among lines differing in respect of the two major allozyme classes EST6-F and EST6-S, despite other evidence that these two classes are not selectively equivalent in the field. However, differences in reproductive fitness were found among lines differing in the minor mobility variants that segregate within EST6-F and EST6-S. A failure to distinguish among these minor forms may explain the discrepancies in previous studies on the effects of the major EST6 allozymes on reproductive fitness. The most significant associations we have found between EST6 and reproductive fitness were due to variation in EST6 activity levels. Male EST6 activity levels were found to be positively correlated with their time to first mating, negatively correlated with the numbers of eggs laid and progeny produced by their mates, and negatively correlated with the frequency with which their mates remate. We conclude that some EST6 variants differ in components of male reproductive fitness operative in laboratory cultures. However, the evidence for fitness differences is stronger for variants affecting the amount, rather than the structure of the enzyme, and the direction of the differences varies between some of the fitness components tested.

Insecticide Resistance as a Model System for Studying Molecular Evolution

Apart from its applied significance, insecticide resistance is an excellent model system for studying the molecular basis of evolutionary change, in particular, the acquisition of a qualitatively different phenotype. It also has the unusual advantage in evolutionary biology that the change has been widespread and rapid enough to be amenable to analysis; approximately 450 species of insects and mites have developed resistance to chemical insecticides over the past forty years (Georghiou, 1986).

Effects of the residue adjacent to the reactive serine on the substrate interactions ofDrosophila esterase 6

Esterase 6 fromDrosophila melanogaster is a carboxylesterase that belongs to the serine esterase multigene family. It has a basic histidine (His) at residue 187, adjacent to the reactive serine (Ser) at residue 188, whereas most other characterized members of the family have an acidic glutamate (Glu) in the equivalent position. We have used site-directedin vitro mutagenesis to replace the His codon of the esterase 6 gene with either Gln or Glu codons. The enzymes encoded by these active-site mutants and a wild-type control have been expressed, purified, and characterized. Substitution of Gln for His at position 187 has little effect on the biochemical properties of esterase 6, but the presence of Glu at this position is associated with three major differences. First, the pH optimum is increased from 7 to 9. Second, the mutant enzyme shows decreased activity for β-naphthyl esters andp-nitrophenyl acetate but has gained the ability to hydrolyze acetylthiocholine. Finally, the Gibbs free energy of activation for the enzyme is increased. These results suggest that residue 187 interacts directly with the substrate alkyl group and that this interaction is fully realized in the transition state. We further propose that the presence of His rather than Glu at position 187 in esterase 6 contributes significantly to its functional divergence from the cholinesterases and that this divergence is due to different interactions between residue 187 and the substrate alkyl group.

Causes and consequences of esterase 6 enzyme activity variation in pre-adult Drosophila melanogaster

We report heritable threefold differences in both larval and pupal esterase 6 activity among 17 isoallelic lines of D. melanogaster extracted from a natural population. The activity differences in the two stages are only weakly correlated with each other or with previously determined values for esterase 6 activity in adults of these lines. The pre-adult activity variation is also unrelated to polymorphisms among the lines for six esterase 6 allozymes and six restriction sites in a region encompassing the esterase 6 coding DNA and the first kbp of 5′ flanking DNA. However, two insertions, of 8.0 and 6.8 kbp, located about 1.4 kbp 5′ of the esterase 6 coding region are associated with low activity in larvae and, to a lesser extent, in pupae, albeit not in adults. Restriction mapping reveals similarity between the 8.0 kbp insert and the 7.4 kbp retrotransposon 17.6. The differences in larval activity among lines are positively correlated with fitness as assessed from assays of pre-adult viability and development time but no significant associations between pupal esterase 6 activity and these measures are detected. Some effects of esterase 6 allozyme differences are also found for viability and development time but these effects could be explained by linkage disequilibrium between the 8.0 kbp insert and the EST6-9 allozyme.