title: An engineered old yellow enzyme that enables efficient synthesis of (4R,6R)-Actinol in a one-pot reduction system.
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.
authors: 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).
authors: Shoichiro
authors: S
authors: Horita
authors: Michihiko
authors: M
authors: Kataoka
authors: Nahoko
authors: N
authors: Kitamura
authors: Takuya
authors: T
authors: Nakagawa
authors: Takuya
authors: T
authors: Miyakawa
authors: Jun
authors: J
authors: Ohtsuka
authors: Koji
authors: K
authors: Nagata
authors: Sakayu
authors: S
authors: Shimizu
authors: Masaru
authors: M
authors: Tanokura
mesh_terms: D000595:Amino Acid Sequence; D001547:Benzaldehydes / Q000737:chemistry / Q000378:metabolism; D055162:Biocatalysis; D002175:Candida / Q000201:enzymology; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003511:Cyclohexanols / Q000138:chemical synthesis*; D003512:Cyclohexanones / Q000138:chemical synthesis* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D008969:Molecular Sequence Data; D009154:Mutation; D009252:NADPH Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D001547:Benzaldehydes; D003511:Cyclohexanols; D003512:Cyclohexanones; D005656:Fungal Proteins; C476259:ketoisophorone; D009252:NADPH Dehydrogenase; C011483:4-hydroxybenzaldehyde
keywords: X-ray crystallography; biocatalysis; enzyme catalysis; enzymes; old yellow enzyme