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) which encodes a thermostable carboxylesterase belonging to the hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. Based on secondary structure predictions and a secondary structure-driven multiple sequence alignment with remote homologous proteins of known three-dimensional structure, we previously hypothesized for this enzyme the alpha/beta-hydrolase fold typical of several lipases and esterases and identified Ser160, Asp 255 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 structure has been recently elucidated. The model reveals the topological organization of the fold corroborating our predictions. As regarding the active-site residues, Ser160, Asp255 and His285 are located close each other at hydrogen bond distances. The catalytic role of Ser160 as the nucleophilic member of the triad is demonstrated by the [(3)H]diisopropylphosphofluoridate (DFP) active-site labeling and sequencing of a radioactive peptide containing the signature sequence GDSAGG.
Manco
G
G
Istituto di Biochimica delle Proteine ed Enzimologia, Via Marconi 10, 80125 Naples, Italy. manco@dafne.ibpe.na.cnr.it
Camardella
L
L
Febbraio
F
F
Adamo
G
G
Carratore
V
V
Rossi
M
M
D000345:Affinity Labels; D000595:Amino Acid Sequence; D019611:Archaeoglobus fulgidus / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics; D003196:Computer Graphics; D004795:Enzyme Stability; D006358:Hot Temperature; D007531:Isoflurophate / Q000493:pharmacokinetics; D008049:Lipase / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D012694:Serine; D013816:Thermodynamics
D000345:Affinity Labels; D007531:Isoflurophate; D012694:Serine; D002265:Carboxylic Ester Hydrolases; D008049:Lipase