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.

The triterpenoid saponin content difference is associated with the two type oxidosqualene cyclase gene copy numbers of Pulsatilla chinensis and Pulsatilla cernua

“…At present, about 170 OSC genes from plants have been functionally analyzed (Chen et al, 2021;Wang et al, 2021). At the same time, the researchers also carried out targeted mutation to find the key active sites of OSC genes (Souza-Moreira et al, 2016;Ito et al, 2017;Aiba et al, 2018;Suzuki et al, 2019). The key active sites affecting the products of the family members were mainly located in the N-terminal, MXCYC conservative region, and Cterminal (Guo et al, 2022).…”