In the present study, the stereoselectivity of purified lipases from Candida rugosa, Chromobacterium viscosum, Pseudomonas species and Rhizopus arrhizus towards triacylglycerols in comparison to various structural analogs were investigated. Different triacylglycerol analogs with distinct polarities at position sn-2 of the glycerol backbone (1,3-diacy1-2-X-glycerol, where 2-X = 2-acyloxy, 2-alkyloxy, 2-deoxy-2-alkyl, or 2-deoxy-2-phenyl) were synthesized. Substrate hydrophobicity and steric requirement was modified by variation of the alkyl and acyl chain length. Hydrolysis of these substrates demonstrated that minor structural variations at C2 of triacylglycerol strongly affect the stereoselectivity of the lipases tested. It was noteworthy that the variation of substrate structure did not only affect the quantity of stereoselectivity expressed as percentage enantiomeric excess, but also resulted in a reversal of stereopreference in some cases. Replacement of the acylester in position 2 of glycerol by a non-ester-linked aliphatic moiety shifted the preference of Chromobacterium viscosum lipase from sn-3 to sn-1 . Lipases from Chromobacterium viscosum, Pseudomonas species and Rhizopus arrhizus exhibited sn-3 preference with 2-deoxy-2-phenyl analogs, while towards substrates with a 2-deoxy-2-alkyl moiety sn-1 stereobias was recorded. Candida rugosa lipase was rather insensitive to substrate variations concerning the polarity at position 2 of the glycerol backbone. However, variation of the acyl chain length significantly influenced stereoselectivity of this lipase.

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.

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The lipases from Rhizopus and Rhizomucor are members of the family of Mucorales lipases. Although they display high sequence homology, their stereoselectivity toward triradylglycerols~sn-2 substituted triacylglycerols! varies. Four different triradylglycerols were investigated, which were classified into two groups: flexible substrates with rotatable O9-C19 ether or ester bonds adjacent to C2 of glycerol and rigid substrates with a rigid N9-C19 amide bond or a phenyl ring in sn-2. Although Rhizopus lipase shows opposite stereopreference for flexible and rigid substrates~hydrolysis in sn-1 and sn-3, respectively!, Rhizomucor lipase hydrolyzes both groups of triradylglycerols preferably in sn-1. To explain these experimental observations, computer-aided molecular modeling was applied to study the molecular basis of stereoselectivity. A generalized model for both lipases of the Mucorales family highlights the residues mediating stereoselectivity:~1! L258, the C-terminal neighbor of the catalytic histidine, and~2! G266, which is located in a loop contacting the glycerol backbone of a bound substrate. Interactions with triradylglycerol substrates are dominated by van der Waals contacts. Stereoselectivity can be predicted by analyzing the value of a single substrate torsion angle that discriminates between sn-1 and sn-3 stereopreference for all substrates and lipases investigated here. This simple model can be easily applied in enzyme and substrate engineering to predict Mucorales lipase variants and synthetic substrates with desired stereoselectivity.

In a model elaborated earlier to understand and predict the stereopreference ofRhizopus oryzae lipase (ROL) catalyzed hydrolysis of triradylglycerols, we identified the degree of flexibility of the C1′‐X′ bond (X = O for ether, N for amide, C for alkyl, methylene, and a phenylring, respectively) adjacent to C2 of glycerol being responsible for the discrimination of the enantiomers (Kovac et al., Eur. J. Lipid Sci. Technol.2000, 61—72). During catalysis of forward and back reaction — hydrolysis and esterification — in either case the carbonyl carbon of the sn‐1 or sn‐3 fatty acid binds to the active site serine of ROL leading to a covalently bound intermediate, which was simulated in the model. Thus, we assumed that stereoselectivity of ROL in esterification of corresponding 2‐monoradylglycerols with oleic acid in cyclohexane should follow the same model. As predicted by this model 2‐monoradylglycerols with “rigid” phenyl and amide substituents were esterified at thesn‐3 position, and those with “ flexible” ether substituents at thesn‐1 position. However, enantiomeric excess of wild type ROL in esterifying 2‐monaradylglycerols with flexible benzylether and methylene substituents differed by around 50% as compared to hydrolysis experiments with corresponding triradylglyc‐erols. In addition esterification of 2‐monoradylglycerol with flexible ether substituent by ROL L258F/L254F double mutant was essentially non‐selective compared to corresponding triradylglycerol where enantiomeric excess was 58%sn‐1. Whether water activity was a factor determining these discrepancies was investigated for ROL‐ and double mutant enzyme‐catalyzed esterification of the ether and methylene substrates under controlled water activities from 0.02—0.85. In all cases stereoselectivities ob‐served were independent from water activities. In conclusion, the model describing the stereoselective course of aqueous hydrolysis of triradylglycerols catalyzed by ROL in most cases applies to the esterification reaction in organic solvent. Differences in stereoselectivity observed are attributed to reduced possibilities for interaction of 2‐monoradylglycerol substrates with the binding sites of ROL as compared to those of triradylglycerol substrates.

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