An Engineered Old Yellow Enzyme that Enables Efficient Synthesis of (4R,6R)‐Actinol in a One‐Pot Reduction System |
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| Authors: | Dr Shoichiro Horita Prof Dr Michihiko Kataoka Nahoko Kitamura Takuya Nakagawa Dr Takuya Miyakawa Dr Jun Ohtsuka Dr Koji Nagata Prof Dr Sakayu Shimizu Prof Dr Masaru Tanokura |
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| Affiliation: | 1. Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1‐1‐1 Yayoi, Bunkyo‐ku, Tokyo 113‐8657 (Japan);2. Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1‐1 Gakuencho, Naka‐ku, Sakai, Osaka 599‐8531 (Japan);3. Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Oiwake, Kitashirakawa, Sakyo‐ku, Kyoto 606‐8502 (Japan) |
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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. |
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| Keywords: | biocatalysis enzyme catalysis enzymes old yellow enzyme X‐ray crystallography |
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