Molecular Basis for the Enantio- and Diastereoselectivity of Burkholderia cepacia Lipase toward γ-Butyrolactone Primary Alcohols

Burkholderia cepacia lipase (BCL) shows high enantioselectivity toward chiral primary alco- hols, but this enantioselectivity is often unpredicta- ble, especially for substrates that contain an oxygen at the stereocenter. For example, BCL resolves b- substituted-g-acetyloxymethyl-g-butyrolactones (ace- tates of a chiral primary alcohol) by hydrolysis of the acetate, but the enantioselectivity varies with the nature and orientation of the b-alkyl substituent. BCL favors the (R)-primary alcohol when the b- alkyl substituent is hydrogen (E = 30) or trans methyl (E = 38), but the (S)-primary alcohol when it is cis methyl (E = 145). To rationalize this unusual selectiv- ity, we used a combination of experiments to show the importance of polar interactions and modeling to reveal differences in orientations of the enantiomers. Removal of either the lactone carbonyl in the sub- strate or the polar side chains in the enzyme by using a related enzyme without these side chains de- creased the enantioselectivity at least four-fold. Mod- eling revealed that the slow enantiomers do not bind by exchanging the location of two substituents rela- tive to the fast enantiomer. Instead, three substitu- ents remain in the same region, but the fourth sub- stituent, hydrogen, inverts to a new location, like an umbrella in a strong wind. In this orientation the fa- vored stereoisomers have similar shapes, thus ac- counting for the unusual stereoselectivity. The ratio of catalytically productive orientations for the fast vs. slow enantiomers in a molecular dynamic simulation correlated (R 2 = 0.82) with the degree of enantiose- lectivity including the case where the enantioselectiv- ity reversed. Weighting this ratio by the ratio of H- bonds in the polar interaction to account for differ- ent binding strengths improved the correlation with the measured enantioselectivity to R 2 = 0.97. The modeling identifies key interactions responsible for high enantioselectivity in this class of substrates.