title: Sequence of the lid affects activity and specificity of Candida rugosa lipase isoenzymes.
abstract: The fungus Candida rugosa produces multiple lipase isoenzymes (CRLs) with distinct differences in substrate specificity, in particular with regard to selectivity toward the fatty acyl chain length. Moreover, isoform CRL3 displays high activity towards cholesterol esters. Lipase isoenzymes share over 80% sequence identity but diverge in the sequence of the lid, a mobile loop that modulates access to the active site. In the active enzyme conformation, the open lid participates in the substrate-binding site and contributes to substrate recognition. To address the role of the lid in CRL activity and specificity, we substituted the lid sequences from isoenzymes CRL3 and CRL4 in recombinant rCRL1, thus obtaining enzymes differing only in this stretch of residues. Swapping the CRL3 lid was sufficient to confer to CRL1 cholesterol esterase activity. On the other hand, a specific shift in the chain-length specificity was not observed. Chimeric proteins displayed different sensitivity to detergents in the reaction medium.
mesh_terms: D020003:1-Octanol / Q000378:metabolism; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002175:Candida / Q000201:enzymology*; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics / Q000502:physiology; D002788:Cholesterol Esters / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004951:Esterification; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D006868:Hydrolysis; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008066:Lipolysis / Q000187:drug effects; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002787:Sterol Esterase / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000235:genetics; D014050:Toluene / Q000494:pharmacology; D014270:Trifluoroethanol / Q000031:analogs & derivatives / Q000378:metabolism; D014280:Triglycerides / Q000378:metabolism; D014304:Triolein / Q000378:metabolism
chemical_list: D002788:Cholesterol Esters; D003902:Detergents; D005227:Fatty Acids; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D014280:Triglycerides; D014304:Triolein; D014050:Toluene; C021976:cholesteryl linoleate; D014270:Trifluoroethanol; D002787:Sterol Esterase; D008049:Lipase; D020003:1-Octanol; C005830:tributyrin