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

Published online by Cambridge University Press:  01 September 1999

GIUSEPPE MANCO
Affiliation:
Istituto di Biochimica delle Proteine ed Enzimologia, CNR, Via Marconi 10, 80125 Naples, Italy
FERDINANDO FEBBRAIO
Affiliation:
Istituto di Biochimica delle Proteine ed Enzimologia, CNR, Via Marconi 10, 80125 Naples, Italy
ELENA ADINOLFI
Affiliation:
Istituto di Biochimica delle Proteine ed Enzimologia, CNR, Via Marconi 10, 80125 Naples, Italy
MOSÈ ROSSI
Affiliation:
Istituto di Biochimica delle Proteine ed Enzimologia, CNR, Via Marconi 10, 80125 Naples, Italy Università degli Studi di Napoli Federico II, Via Mezzocannone 16, 80100 Naples, Italy
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Abstract

The moderate thermophilic eubacterium Alicyclobacillus (formerly Bacillus) acidocaldarius expresses a thermostable carboxylesterase (esterase 2) belonging to the hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. Based on secondary structures predictions and a secondary structure-driven multiple sequence alignment with remote homologous protein of known three-dimensional (3D) structure, we previously hypothesized for this enzyme the α/β-hydrolase fold typical of several lipases and esterases and identified Ser155, Asp252, and His282 as the putative members of the catalytic triad. In this paper we report the construction of a 3D model for this enzyme based on 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, Ser155, Asp252, and His282 are located close to each other at hydrogen bond distances. Their catalytic role was here probed by biochemical and mutagenic studies. Moreover, on the basis of the secondary structure-driven multiple sequence alignment and the 3D structural model, a residue supposed important for catalysis, Gly84, was mutated to Ser. The activity of the mutated enzyme was drastically reduced. We propose that Gly84 is part of a putative “oxyanion hole” involved in the stabilization of the transition state similar to the C group of the esterase/lipase family.

Type
Research Article
Copyright
© 1999 The Protein Society

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