Homology modeling and identification of serine 160 as nucleophile of the active site in a thermostable carboxylesterase from the archaeon Archaeoglobus fulgidus

The hyperthermophilic Archaeon Archaeoglobus fulgidus has a gene (AF1763) w hich encodes a thermostable carboxylesterase belonging to the hormone-sensi tive lipase (HSL)-like group of the esterase/lipase family, Based on second ary structure predictions and a secondary structure driven multiple sequenc e alignment with remote homologous proteins of known three-dimensional stru cture, we previously hypothesized for this enzyme the alpha/beta-hydrolase fold typical of several lipases and esterases and identified Ser160, Asp 25 5 and His285 as the putative members of the catalytic triad. In this paper we report the building of a 3D model for this enzyme based on the structure of the homologous brefeldin A esterase from Bacillus subtilis whose struct ure has been recently elucidated, The model reveals the topological organiz ation of the fold corroborating our predictions. As regarding the active-si te residues, Ser160, Asp255 and His285 are located close each other at hydr ogen bond distances, The catalytic role of Ser160 as the nucleophilic membe r of the triad is demonstrated by the [H-3]diisopropylphosphofluoridate (DF P) active-site labeling and sequencing of a radioactive peptide containing the signature sequence GDSAGG.