An engineered old yellow enzyme that enables efficient synthesis of (4R,6R)-Actinol in a one-pot reduction system.

Shoichiro Horita, Michihiko Kataoka, Nahoko Kitamura, Takuya Nakagawa, Takuya Miyakawa, Jun Ohtsuka, Koji Nagata, Sakayu Shimizu, Masaru Tanokura

Index: ChemBioChem. 16(3) , 440-5, (2015)

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Abstract

(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.© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.