Biochemical characterization of a GDSL-motif esterase from Bacillus sp. K91 with a new putative catalytic mechanism.

The esterase gene Est8 from the thermophilic bacterium Bacillus sp. K91 was cloned and expressed in Escherichia coli. The monomeric enzyme exhibited a theoretical molecular mass of 24.5 kDa and an optimal activity around 50°C at pH 9.0. A model of Est8 was constructed using a hypothetical YxiM precursor structure (2O14_A) from Bacillus subtilis as template. The structure showed an α/β-hydrolase fold and indicated the presence of a typical catalytic triad consisting of Ser-11, Asp-182, and His-185, which were investigated by site-directed replacements coupled with kinetic characterization. Asp-182 and His-185 residues were more critical than the Ser-11 residue in the catalytic activity of Est8. A comparison of the amino acid sequence showed that Est8 could be grouped into the GDSL family and further classified as an SGNH hydrolase. Est8 is a new member of the SGNH hydrolase subfamily and may employ a different catalytic mechanism.