Stereoselectivity of mucorales lipases toward triradylglycerols—A simple solution to a complex problem

Abstract

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.

Improvement of Yarrowia lipolytica Lipase Enantioselectivity by Using Mutagenesis Targeted to the Substrate Binding Site

“…The hydrophobic crevice consists of T88, V94, I98, I100, F129, L163, P190, V232, V235, P236, Y241 corresponding to S82, W88, L92, F94, F111, L145, P177, V205, L208, P209 and F215 in R. miehei lipase. [21] The scissile fatty acid of a triglyceride is supposed to bind to this hydrophobic crevice. The sn-2 substituent would bind to the hydrophobic dent formed by I204, T252, V254 and L258 in R. miehei lipase and by I231, V283, V285 and L290.…”

“…The sn-2 substituent would bind to the hydrophobic dent formed by I204, T252, V254 and L258 in R. miehei lipase and by I231, V283, V285 and L290. [21] Figure 4 represents the superimposition of Lip2p and lipases from Rhizomucor miehei and Thermomyces lanuginosa lipases.…”

Semiempirical Molecular Orbital Studies of the Acylation Step in the Lipase‐Catalyzed Ester Hydrolysis

“…A further slight decrease in d 6 was observed when TI1 was formed, which was accompanied by proton transfer from Ser to His, with d 5 and d 4 being lengthened and shortened, respectively. At TI1, the proton has been transferred to His as can be seen from d 4 …”

“…Some important geometrical parameters for the stationary points during the catalytic reaction are summarized in Table 2. The most representative bond lengths are probably d 4 , d 5 and d 6 . It is obvious that for all models studied here, the locations of TS1 and TI1 were almost the same.…”