Rational design of Rhizopus oryzae lipase with modified stereoselectivity toward triradylglycerols

Abstract

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.

A Molecular Mechanism of Enantiorecognition of Tertiary Alcohols by Carboxylesterases

“…[10,15,19,20,34] The results for secondary alcohols are in accordance with complementary methods which model enantioselectivity in hydrolasecatalyzed reactions by evaluating the free energy of enzyme-substrate complexes. [21,35] Small changes in structure lead to small changes in enantioselectivity Intuitively, one would expect that homologous enzymes have identical enantiopreference as it was observed for…”

“…by switching from L-to D-tartrate in the Sharpless-epoxidation [39] by a simple and general rule. [16,17] Changes of enantiopreference by point mutations in the substrate binding site have also been observed for lipases, [15] phosphotriesterases, [41] lactate dehydrogenases, [42] and alcohol dehydrogenases. [43] Thus, our results support the general idea that enantiopreference is not an inherent property of an enzyme but can be tuned by protein engineering.…”

“…Several attempts to vary enantioselectivity of esterases and lipases by directed evolution, [11][12][13] and by rational protein engineering were successful. [14,15] The latter method also yields insight into the molecular mechanism of enantiorecognition. X-ray structures of several lipases and esterases complexed with chiral substrate analogous inhibitors are available.…”

Structural basis of stereoselectivity in Candida rugosa lipase-catalyzed hydrolysis of secondary alcohols

“…As shown previously, docking methods are generally reliable and able to correctly predict stereopreference [5,22,23]. However, problems occur when energy-based scoring strategies are used to rank substrates [24].…”

Molecular basis of lipase stereoselectivity

“…This hypothesis was tested replacing Leu258 either by the smaller amino acids alanine and serine or by the bulky phenylalanine. According to the model, the ROL Leu258Phe exhibited an increased sn-3 stereoselectivity toward triacylglycerol analogs with rigid and/or bulky sn-2 substituents adjacent to C2 of glycerol, while Leu258Ala and Leu258Ser substitutions decreased the sn-3 stereoselectivity or even reversed it (see section 6 for more details) [71]. Combining both rigidity and bulkiness of the sn-2 substituent, hydrolysis of ROL Leu258Phe toward the benzoylamide substrate revealed the most significant increase in stereoselectivity as compared to ROL wild type (E was 195 and 15, respectively, at 50 % conversion; Fig.…”