Identification of Residues Essential for Differential Fatty Acyl Specificity of Geotrichum candidum Lipases I and II

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Abstract
The fungus Geotrichum candidum produces two lipase isoenzymes, GCL I and GCL II, with distinct differences in substrate specificity despite their 86% identical primary structure. GCL I prefers ester substrates with long-chain cis (delta-9) unsaturated fatty acid moieties, whereas GCL II also accepts medium-length (C8-C14) acyl moieties in the substrate. To reveal structural elements responsible for differences in substrate differentiating ability of these isoenzymes, we designed, expressed, and characterized 12 recombinant lipase variants. Three chimeric lipases containing unique portions of the N-terminal and the C-terminal part of GCL I and GCL II, respectively, were constructed and enzymatically characterized. Activities were measured against mixed triglyceride-poly(dimethyl siloxane) particles. Our results indicate that residues within sequence positions 349-406 are essential for GCL I's high triolein/trioctanoin activity ratio of 20. The substitution of that segment in the specific GCL I to the corresponding residues in the nonspecific GCL II resulted in an enzyme with a triolein/trioctanoin activity ratio of 1.4, identical to that of GCL II. The reverse mutation in GCL II increased its specificity for triolein by a factor of 2, thus only in part restoring the high specificity seen with GCL I. In further experiments, the point mutations at the active site entrance of the GCL I, Leu358Phe and Ile357Ala/Leu358Phe, lowered the triolein/trioctanoin activity ratio from 20 to 4 and 2.5, respectively. The substitutions Cys379Phe/Ser380Tyr at the bottom of the active site cavity of GCL I decreased its specificity to a value of 3.6. Measurements of lipase activity with substrate particles composed of pure triglycerides or ethyl esters of oleic and octanoic acids resulted in qualitatively similar results as reported above. Our data reveal for the first time the identity of residues essential for the unusual substrate preference of GCL I and show that the anatomy, both at the entrance and the bottom of the active site cavity, plays a key role in substrate discrimination.