title: Loop-Grafted Old Yellow Enzymes in the Bienzymatic Cascade Reduction of Allylic Alcohols.
abstract: The enzymatic reduction of C=C bonds in allylic alcohols with Old Yellow Enzymes represents a challenging task, due to insufficient activation through the hydroxy group. In our work, we coupled an alcohol dehydrogenase with three wild-type ene reductases-namely nicotinamide-dependent cyclohex-2-en-1-one reductase (NCR) from Zymomonas mobilis, OYE1 from Saccharomyces pastorianus and morphinone reductase (MR) from Pseudomonas putida M10-and four rationally designed β/α loop variants of NCR in the bienzymatic cascade hydrogenation of allylic alcohols. Remarkably, the wild type of NCR was not able to catalyse the cascade reaction whereas MR and OYE1 demonstrated high to excellent activities. Through the rational loop grafting of two intrinsic β/α surface loop regions near the entrance of the active site of NCR with the corresponding loops from OYE1 or MR we successfully transferred the cascade reduction activity from one family member to another. Further we observed that loop grafting revealed certain influences on the interaction with the nicotinamide cofactor.
authors.affiliation: Institute of Technical Biochemistry, Universität Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Reich
authors.affiliation: Institute of Technical Biochemistry, Universität Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
authors.forename: Bettina M
authors.initials: BM
authors.lastname: Nestl
authors.affiliation: Institute of Technical Biochemistry, Universität Stuttgart, Allmandring 31, 70569, Stuttgart, Germany. bernhard.hauer@itb.uni-stuttgart.de.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Hauer
mesh_terms: D000426:Alcohol Dehydrogenase / Q000378:metabolism; D000595:Amino Acid Sequence; D008958:Models, Molecular; D015394:Molecular Structure; D009252:NADPH Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D020005:Propanols / Q000737:chemistry* / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D020005:Propanols; C006463:allyl alcohol; D000426:Alcohol Dehydrogenase; D009252:NADPH Dehydrogenase; C020722:cinnamyl alcohol
keywords: allylic alcohols; biocatalysis; cascade reactions; loop grafting; oxidoreductases; reduction