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.