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Abstract

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.

Molecular basis for the behavioral effects of the odorant degrading enzyme Esterase 6 in Drosophila

“…After six rounds of directed evolution, the EST6 variant with greatest soluble expression (EST6-1) contained 16 mutations; K15V, V145L, R208K, G229E, N237S, T247A, D290G, I292F, I335V, E383G, S400G, A416V, F450S, F456S, N485D, I511T (note that amino acids are numbered from the first residue of the mature EST6 protein as it would be processed in its native form within the fly33 and omits the start methionine included to permit heterologous expression in E. coli ). Four of these mutations have been found in EST6 from several Drosophila species (V145A, R208K, T247A and I292F)2334. Importantly, the catalytic activity of EST6-1 was very similar to that of EST6-WT (Supplementary Fig.…”

“…However, the juvenile hormone esterase from the moth Manduca sexta (Ms JHE)20, an insecticide metabolizing carboxylesterase from the blowfly Lucilia cuprina (Lc αE7)21 and acetylcholinesterase from D. melanogaster (Dm AChE)22 are the only insect CCEs for which crystal structures have been determined, so relatively little is known of the structure-function relationships underlying their diverse functions. The structural features of EST6 have so far been inferred from the structure of the D. melanogaster AChE or its orthologue from the electric ray Torpedo californica , but the low sequence similarity between EST6 and AChE (27%) means that the fine structural features of the enzyme responsible for its substrate specificity have not yet been understood23.…”