Oryza sativa Parkeol Cyclase: Changes in the Substrate-Folding Conformation and the Deprotonation Sites on Mutation at Tyr257: Importance of the Hydroxy Group and Steric Bulk

Abstract

Y257 of Oryza sativa parkeol synthase (OsOSC2) corresponds to H234 of Saccharomyces cerevisiae lanosterol cyclase (ScLAS), which is believed to be responsible for the final deprotonation reaction. An Ala mutant afforded nine tetracyclic skeletons as the main products; they consisted of protostadienol scaffolds with both 17R and 17S configurations and both 20R and 20S configurations, as well as a pair of 20R- and 20S-configured parkeols. The production of 20R- and 20S-configured tetracycles (59:40 ratio) through the action of the Y257A mutant indicated that the substrate folding had been altered from a chair-boat-chair-chair (a normal folding pattern) to a chair-boat-chair-boat structure (an unusual folding pattern). Other mutants with different steric bulks also yielded both 20R- and 20S-configured tetracycles. Thus, the primary function of Y257 appears to be to impose a normal chair structure at the D-ring site through having appropriate steric bulk. In contrast, mutations at H234 of ScLAS were reported to cause no conformational changes. The OsOSC2 Phe mutant also yielded 20R- and 20S-configured parkeols (25:33 ratio), thus suggesting that the OH group of Y257 can form hydrogen bonds with other amino acids to force a chair conformation at the D-ring site, and this variant also gave 20R- and 20S-configured parkeols in a high yield (60 %). Y257 is unlikely to act as a base to abstract H-11 and stabilize the transient cation through cation-π interactions. Thus, the catalytic roles of Y257 are significantly different from those of H234 of ScLAS.

Keywords: enzyme catalysis; lanosterol; oxidosqualene cyclases; parkeol; terpenoids.