RATIONAL DESIGN OF RHIZOPUS-ORYZAE LIPASE WITH MODIFIED STEREOSELECTIVITY TOWARD TRIRADYLGLYCEROLS

The binding site of sn-1(3)-regioselective Rhizopus oryzae lipase (ROL ) has been engineered to change the stereoselectivity of hydrolysis of triacylglycerol substrates and analogs. Two types of prochiral trirad ylglycerols were considered: 'flexible' substrates with ether, benzyle ther or ester groups, and 'rigid' substrates with amide or phenyl grou ps, respectively, in the sn-2 position. The molecular basis of sn-1(3) stereoselectivity of ROL was investigated by modeling the interaction s between substrates and ROL, and the model was confirmed by experimen tal determination of the stereoselectivity of wild-type and mutated RO L, For the substrates, the following rules were derived: (i) stereo-pr eference of ROL toward triradylglycerols depends on the substrate stru cture. Substrates with 'flexible' sn-2 substituents are preferably hyd rolyzed at sn-1, 'rigid' substrates at sn-3. (ii) Stereopreference of ROL toward triradylglycerols can be predicted by analyzing the geometr y of the substrate docked to ROL: if the torsion angle Phi(O3-C3) Of g lycerol is more than 150 degrees, the substrate will preferably be hyd rolyzed in sn-l, otherwise in sn-3, For ROL, the following rules were derived: (i) residue 258 affects stereoselectivity by steric interacti ons with the sn-2 substituent rather than polar interactions, To a low er extent, stereoselectivity is influenced by mutations further apart (L254) from residue 258, (ii) With 'rigid' substrates, increasing the size of the binding site (mutations L258A and L258S) shifts stereosele ctivity of hydrolysis toward sn-1, decreasing its size (L258F and L258 F/L254F) toward sn-3.