Scheib H, Pleiss J, Stadler P, Kovac A, Potthoff A P, Haalck L, Spener F, Paltauf F, Schmid R D
Institute of Technical Biochemistry, University of Stuttgart, Germany.
Protein Eng. 1998 Aug;11(8):675-82. doi: 10.1093/protein/11.8.675.
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 triradylglycerols were considered: 'flexible' substrates with ether, benzylether or ester groups, and 'rigid' substrates with amide or phenyl groups, respectively, in the sn-2 position. The molecular basis of sn-1(3) stereoselectivity of ROL was investigated by modeling the interactions between substrates and ROL, and the model was confirmed by experimental determination of the stereoselectivity of wild-type and mutated ROL. For the substrates, the following rules were derived: (i) stereopreference of ROL toward triradylglycerols depends on the substrate structure. Substrates with 'flexible' sn-2 substituents are preferably hydrolyzed at sn-1, 'rigid' substrates at sn-3. (ii) Stereopreference of ROL toward triradylglycerols can be predicted by analyzing the geometry of the substrate docked to ROL: if the torsion angle phiO3-C3 of glycerol is more than 150 degrees, the substrate will preferably be hydrolyzed in sn-1, otherwise in sn-3. For ROL, the following rules were derived: (i) residue 258 affects stereoselectivity by steric interactions with the sn-2 substituent rather than polar interactions. To a lower 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 stereoselectivity of hydrolysis toward sn-1, decreasing its size (L258F and L258F/L254F) toward sn-3.
米根霉脂肪酶（ROL）的sn-1(3)-区域选择性结合位点已被改造，以改变三酰甘油底物及类似物的水解立体选择性。研究了两种前手性三酰甘油：分别在sn-2位带有醚基、苄醚基或酯基的“柔性”底物，以及带有酰胺基或苯基的“刚性”底物。通过模拟底物与ROL之间的相互作用，研究了ROL的sn-1(3)立体选择性的分子基础，并通过对野生型和突变型ROL的立体选择性进行实验测定，证实了该模型。对于底物，得出了以下规律：（i）ROL对三酰甘油的立体偏好取决于底物结构。具有“柔性”sn-2取代基的底物优先在sn-1位水解，“刚性”底物在sn-3位水解。（ii）通过分析对接至ROL的底物的几何结构，可以预测ROL对三酰甘油的立体偏好：如果甘油的扭转角phiO3-C3大于150度，底物将优先在sn-1位水解，否则在sn-3位水解。对于ROL，得出了以下规律：（i）残基258通过与sn-2取代基的空间相互作用而非极性相互作用影响立体选择性。在较低程度上，立体选择性受到与残基258距离更远的突变（L254）的影响。（ii）对于“刚性”底物，增大结合位点的尺寸（突变L258A和L258S）会使水解的立体选择性向sn-1位偏移，减小其尺寸（L258F和L258F/L254F）则向sn-3位偏移。
