5.4 Modeling and engineering of stereoselectivity
To date, there is no general rule to explain thesn-1(3) stereopreference of all lipases
toward triacylglycerol andsn-2 substituted analogs. It has been shown experimen-
tally that stereopreference and stereoselectivity toward triacylglycerol and analogs
depend on both substrate structure and the type of lipase used as catalyst (Paltauf and
Wagner, 1976; Ransac et al., 1990; Rogalska et al., 1993; 1995; Stadler et al., 1995;
Kovac et al., 1996). This is in contrast to lipase stereopreference toward chiral sec-
ondary alcohols, where all lipases prefer the same enantiomer. A rule to predict the
preferred enantiomer has been proposed which is based exclusively on structure of
the secondary alcohol (Kazlauskas et al., 1991). Generalization of this rule to pri-
mary alcohols was restricted to alcohols without oxygen bound at the stereocenter
(Weissfloch and Kazlauskas, 1995); thus, it is not applicable to triacylglycerols. For
Mucoraleslipases, a simple method to explain and predict stereoselectivity toward
this type of substrates has been proposed (Haalck et al., 1997; Holzwarth et al., 1997;
Scheib et al., 1998; 1999). It is based on docking of the substrate to the binding site of
Rhizopus oryzaelipase (ROL) or RML, and uses molecular dynamics simulations to
relax both substrate and protein side chains. Two hydrophobic patches in the binding
site of ROL and RML were identified to which the acyl chains of the substrate were
assumed to bind: the deephydrophobic creviceconsists of the amino acids T83, A89,
I93, F95, F112, L146, P178, V206, V209, P210, and F216 (numbering in ROL), and
the shallowhydrophobic dentwhich comprises I205, T252, L254, and L258. Each
substrate was docked in two orientations: insn-1 orientation, where thesn-1 fatty
92 5 Molecular Basis of Specificity and Stereoselectivity of Microbial Lipases
Figure 4. Left: stereoselective hydrolysis of triacylglycerols and analogs tosn-1,2- orsn-2,3-diacyl-
glycerols and analogs (Scheib et al., 1998). Right:sn-2 substituents attached to the (pro-) chiral C2 of
glycerol with trivial (top) and systematic (bottom) substrate names. Ether and ester substrates are classi-
fied as ‘flexible’, amide and phenyl substrates as ‘rigid’.