National Center for
4TMC: Crystal Structure Of Old Yellow Enzyme From Candida Macedoniensis Aku4588 Complexed With P-hydroxybenzaldehyde
An Engineered Old Yellow Enzyme that Enables Efficient Synthesis of (4R,6R)-Actinol in a One-Pot Reduction System
Chembiochem (2015) 16 p.440-445
(4R,6R)-Actinol can be stereo-selectively synthesized from ketoisophorone by a two-step conversion using a mixture of two enzymes: Candida macedoniensis old yellow enzyme (CmOYE) and Corynebacterium aquaticum (6R)-levodione reductase. However, (4S)-phorenol, an intermediate, accumulates because of the limited substrate range of CmOYE. To address this issue, we solved crystal structures of CmOYE in the presence and absence of a substrate analogue p-HBA, and introduced point mutations into the substrate-recognition loop. The most effective mutant (P295G) showed two- and 12-fold higher catalytic activities toward ketoisophorone and (4S)-phorenol, respectively, than the wild-type, and improved the yield of the two-step conversion from 67.2 to 90.1 %. Our results demonstrate that the substrate range of an enzyme can be changed by introducing mutation(s) into a substrate-recognition loop. This method can be applied to the development of other favorable OYEs with different substrate preferences.