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The use of alkene reductases for stereoselective C=C bond reduction has become an increasingly useful synthetic method.Flavoprotein alkene reductases such as Saccharomyces pastorianus Old Yellow Enzyme (OYE 1) show very high stereoselectivities; however,only one product enantiomer can be obtained from the wild-type enzyme and their substrate range may be limited.We used the enantiomers of carvone as test substrates to uncover structure-function relationships in S.pastorianus OYE 1.We initially targeted Trp 116,which forms one wall of the substrate binding pocket.Surprisingly,very similar amino acid substitutions,e.g.,Val,Ile and Leu,produced variants whose stereoselectivities differed widely from the wild-type and from one another.We also studied a second reaction catalyzed by OYE 1,the dismutation of carvone to the corresponding phenol as a probe of active site geometry.Finally,to understand exactly how these mutations impacted stereoselectivity,we solved the x-ray crystal structures for the wild-type and all key variants,both in free form and with bound substrates and / or inhibitors.The combined data have shown how very small changes at a critical position in OYE 1 lead to very diverse stereoselectivities.Our future goal is to use this information to extend these stereochemical changes to other substrate classes.