Pancreatic carboxylester lipase from Atlantic salmon (Salmo salar). cDNA sequence and computer-assisted modelling of tertiary structure.
We report the isolation and characterization of a 1795-bp cDNA fragment encoding Atlantic salmon pancreatic carboxylester lipase from salmon pancreas mRNA. The nearly full-length cDNA contained a 540-amino-acid open-reading frame, encompassing the mature protein (by similarity to mammalian carboxylester lipase enzymes). The salmon carboxylester lipase primary structure shared 58% identity with mammalian carboxylester lipases, lacking the proline-rich C-terminal repeats found in human and rat carboxylester lipases. Congruent with other esterase B type enzymes, the salmon carboxylester lipase contained a canonical serine-esterase catalytic triad motif consisting of serine, histidine and aspartic acid. Computer-assisted modelling of the tertiary structure for salmon carboxylester lipase was conducted using acetylcholine esterase (Torpedo californica) as a template structure. The model, in conjunction with sequence comparisons and available enzymological data, has been used to locate putative bile-salt-binding and lipid-binding sites. The carboxylester lipase enzymes contain a unique, highly conserved insert region that may be associated with bile-salt binding. In the model structure, this region is located close to the active site, and contains a tyrosine residue with an adjacent carboxylester-lipase-conserved arginine. These traits have previously been predicted for the non-specific (regarding bile-salt hydroxylation) bile-salt-binding site in carboxylester lipase enzymes. At this site, a dihydroxy or trihydroxy bile-salt molecule may bind the tyrosine via hydrophobic interactions, the anionic bile-salt head group may bind the arginine, while hydrogen bonding between the bile-salt 12 alpha hydroxy group and an adjacent aspargine residue is possible. The model does not contain an active site 'lid' structure as found in other lipases. The carboxylester lipase structural homolog to the 'flap' of the lipases from Geotrichum candidum and Candida rugosa contains a carboxylester-lipase-conserved deletion that renders this region unable to cover the active site. Instead, the shortening of this loop leads to solvent exposure of the carboxylester lipase insert region, an additional indication of the functional importance of this region.
Gjellesvik
D R
DR
Laboratory for Marine Molecular Biology, University of Bergen, Norway.
Lorens
J B
JB
Male
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001647:Bile Acids and Salts / Q000378:metabolism; D001665:Binding Sites; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics*; D003198:Computer Simulation; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000737:chemistry*; D050356:Lipid Metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010179:Pancreas / Q000201:enzymology*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012474:Salmon; D016415:Sequence Alignment
D001647:Bile Acids and Salts; D018076:DNA, Complementary; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase; D003545:Cysteine