Homology modeling and active-site residues probing of the thermophilic Alicyclobacillus acidocaldarius esterase 2

Citation
G. Manco et al., Homology modeling and active-site residues probing of the thermophilic Alicyclobacillus acidocaldarius esterase 2, PROTEIN SCI, 8(9), 1999, pp. 1789-1796
Citations number
35
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
PROTEIN SCIENCE
ISSN journal
0961-8368 → ACNP
Volume
8
Issue
9
Year of publication
1999
Pages
1789 - 1796
Database
ISI
SICI code
0961-8368(199909)8:9<1789:HMAARP>2.0.ZU;2-6
Abstract
The moderate thermophilic eubacterium Alicyclobacillus (formerly Bacillus) acidocaldarius expresses a thermostable carboxylesterase (esterase 2) belon ging to the hormone-sensitive lipase (HSL)-like group of the esterase/lipas e family. Based on secondary structures predictions and a secondary structu re-driven multiple sequence alignment with remote homologous protein of kno wn three-dimensional (3D) structure, we previously hypothesized for this en zyme the alpha/beta-hydrolase fold typical of several lipases and esterases and identified Ser155, Asp252, and His282 as the putative members of the c atalytic triad. In this paper we report the construction of a 3D model for this enzyme based an the structure of mouse acetylcholinesterase complexed with fasciculin. The model reveals the topological organization of the fold corroborating our predictions. As regarding the active-site residues, Ser1 55, Asp252, and His282 are located close to each other at hydrogen bond dis tances. Their catalytic role was here probed by biochemical and mutagenic s tudies. Moreover, on the basis of the secondary structure-driven multiple s equence alignment and the 3D structural model, a residue supposed important for catalysis, Gly84, was mutated to Ser. The activity of the mutated enzy me was drastically reduced. We propose that Gly84 is part of a putative "ox yanion hole" involved in the stabilization of the transition state similar to the C group of the esterase/lipase family.