Over 30 carboxylester hydrolases have been identified in D. melanogaster. Most are classified as acetyl, carboxyl or cholinesterases. Sequence similarities among most of the carboxyl and all the cholinesterases so far characterised from D. 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/beta hydrolases. Fifteen esterase genes have been mapped in D. melanogaster and twelve are clustered at two chromosomal sites. The constitution of each cluster varies across Drosophila 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 in D. melanogaster. These polymorphisms are associated with differences in substrate interactions in vitro and demographic data indicate that the alternative forms are not selectively equivalent in vivo.

In most lineages of the subgenus Sophophora esterase-6 is a homodimeric haemolymph protein. In the melanogaster subgroup of species it has become a monomer which is mainly expressed in the male sperm ejaculatory duct. Our analyses of esterase-6 sequences from three melanogaster subgroup species and two close relatives reveal a brief period of accelerated amino acid sequence change during the transition between the ancestral and derived states. In this period of 2-6Myr the ratio of replacement to silent site substitutions (0.51) is about three times higher than the values in other lineages of the phylogeny. There are about 50 more replacements in this period than would be predicted from the ratios of replacement to silent site substitutions found elsewhere in the phylogeny. Modelling on the known structure of a related acetylcholinesterase suggests that an unusually high proportion of the replacements in the transitional branch are non-conservative changes on the protein surface. Up to half the accelerated replacement rate can be accounted for by clusters of changes to the face of the molecule containing the opening of the active site gorge. This includes changes in and around regions homologous to peripheral substrate binding sites in acetylcholinesterase. There are also three changes in glycosylation status. One region predicted to lie on the protein surface which becomes markedly more hydrophilic is proposed to be the ancestral dimerisation site that is lost in the transitional branch.

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