Zoological Studies

Vol. 53, 2014

Purification and characterization of a specific late-larval esterase from two species of the Drosophila repleta group: contributions to understand its evolution

Vanessa F Lopes1, Hamilton Cabral2, Luciana PB Machado1 and Rogério P Mateus1*

1Laboratório de Genética e Evolução, Departamento de Ciências Biológicas, Universidade Estadual do Centro-Oeste (UNICENTRO), Campus CEDETEG, R. Simeão Camargo Varela de Sá, 03–Vila Carli, Guarapuava, Paraná 85040-080, Brazil
2Laboratório de Tecnologia Enzimática, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo (USP), Av. do Café - s/n, Bairro Monte Alegre, Ribeirão, Preto, São Paulo 14040-903, Brazil

Background: After duplication, one copy of an original gene can become redundant and decay toward a pseudogene status or functionally diverge. Here, we performed the purification and biochemical characterization of EST-4 (a late larval β-esterase) from two Drosophila repleta group species, Drosophila mulleri and Drosophila arizonae, in order to establish comparative parameters between these enzymes in these species and to contribute to better understand their evolution.
In D. mulleri, EST-4 had an optimal activity in temperatures ranging from 40° to 45°C and at pH 7.5, maintaining stability in alkaline pH (8.0 to 10.0). It was classified as serine esterase as its activity was inhibited by PMSF. No ion negatively modulated EST-4 activity, and iron had the most positive modulating effect. In D. arizonae, it showed similar optimum temperature (40°C), pH (8.0), and was also classified as a serine esterase, but the enzymatic stability was maintained in an acidic pH (5.5 to 6.5). Fe+2 had the opposite effect found in D. mulleri, that is, negative modulation. Al+3 almost totally inhibited the EST-4 activity, and Na+ and Cu+2 had a positive modulation effect. Kinetic studies, using ρ-nitrophenyl acetate as substrate, showed that EST-4 from D. mulleri had higher affinity, while in D. arizonae, it showed higher Vmax and catalytic efficiency in optimal reaction conditions.
Conclusions: EST-4 from D. mulleri and D. arizonae are very closely related and still maintain several similar features; however, they show some degree of differentiation. Considering that EST-4 from D. mulleri has more conspicuous gel mobility difference among all EST-4 studied so far and a lower catalytic efficiency was observed here, we proposed that after duplication, this new copy of the original gene became redundant and started to decay toward a pseudogene status in this species, which probably is not occurring in D. arizonae.

Key words: β-esterase; EST-4; D. mulleri; D. arizonae; Evolutionary biochemistry.

*Correspondence: E-mail: rogeriopmateus@gmail.com