Rational design of Rhizopus oryzae lipase with modified stereoselectivity toward triradylglycerols |
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Authors: | Scheib H; Pleiss J; Stadler P; Kovac A; Potthoff AP; Haalck L; Spener F; Paltauf F; Schmid RD |
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Affiliation: | Institute of Technical Biochemistry, University of Stuttgart, Germany. |
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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.
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