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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
Abstract
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.
Results: 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
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