Rational design of Rhizopus oryzae lipase with modified stereoselectivity toward triradylglycerols

The binding site of sn-1(3)-regioselective Rhizopus oryzae lipase (ROL) hasbeen engineered to change the stereoselectivity of hydrolysis oftriacylglycerol substrates and analogs. Two types of prochiraltriradylglycerols were considered: 'flexible' substrates with ether,benzylether or ester groups, and 'rigid' substrates with amide or phenylgroups, respectively, in the sn-2 position. The molecular basis of sn-1(3)stereoselectivity of ROL was investigated by modeling the interactionsbetween substrates and ROL, and the model was confirmed by experimentaldetermination of the stereoselectivity of wild-type and mutated ROL. Forthe substrates, the following rules were derived: (i) stereopreference ofROL toward triradylglycerols depends on the substrate structure. Substrateswith 'flexible' sn-2 substituents are preferably hydrolyzed at sn-1,'rigid' substrates at sn-3. (ii) Stereopreference of ROL towardtriradylglycerols can be predicted by analyzing the geometry of thesubstrate docked to ROL: if the torsion angle phiO3-C3 of glycerol is morethan 150 degrees, the substrate will preferably be hydrolyzed in sn-1,otherwise in sn-3. For ROL, the following rules were derived: (i) residue258 affects stereoselectivity by steric interactions with the sn-2substituent rather than polar interactions. To a lower extent,stereoselectivity is influenced by mutations further apart (L254) fromresidue 258. (ii) With 'rigid' substrates, increasing the size of thebinding site (mutations L258A and L258S) shifts stereoselectivity ofhydrolysis toward sn-1, decreasing its size (L258F and L258F/L254F) towardsn-3.