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An Engineered Old Yellow Enzyme that Enables Efficient Synthesis of (4 R ,6 R )‐Actinol in a One‐Pot Reduction System
Author(s) -
Horita Shoichiro,
Kataoka Michihiko,
Kitamura Nahoko,
Nakagawa Takuya,
Miyakawa Takuya,
Ohtsuka Jun,
Nagata Koji,
Shimizu Sakayu,
Tanokura Masaru
Publication year - 2015
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201402555
Subject(s) - substrate (aquarium) , mutant , enzyme , yield (engineering) , reductase , biocatalysis , chemistry , stereochemistry , protein engineering , combinatorial chemistry , catalysis , biochemistry , materials science , biology , reaction mechanism , metallurgy , ecology , gene
(4 R ,6 R )‐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 (6 R )‐levodione reductase. However, (4 S )‐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 (4 S )‐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.