LINKING DIVERSITY IN EVOLUTIONARY ORIGIN AND STEREOSPECIFICITY FOR ENOYL THIOESTER REDUCTASES - DETERMINATION AND INTERPRETATION OF THE NOVEL STEREOCHEMICAL COURSE OF REACTION CATALYZED BY CROTONYL COA REDUCTASE FROM STREPTOMYCES-COLLINUS

The stereochemical course of reduction of crotonyl CoA by the novel cr otonyl CoA reductase (CCR) of Streptomyces collinus was determined usi ng a radiochemical assay. The reaction was shown to proceed with trans fer of the hydrogen from the pro-4S position of NADPH to the Re face o f the beta-carbon of crotonyl CoA. This transfer represents the first exception to the observation that enoyl thioester reductases catalyze transfer of the pro-4S hydrogen of NADPH to the Si face of the substra te. The observation of addition of solvent hydrogen to the Re face of the alpha-carbon in the reaction catalyzed by CCR demonstrated that th e overall reduction of crotonyl CoA proceeds in an anti fashion. The o verall stereochemical outcome of the reaction catalyzed by CCR is diff erent to the four stereochemical outcomes that have previously been ob served for enoyl thioester reductases. It is significant that the pred icted amino acid sequence of CCR has also been shown to be unrelated t o other enoyl thioester reductases. Based on these observations it is proposed that the stereochemical course of an enoyl thioester reductio n serves no mechanistic function but merely reflects the pedigree or a ncestral lineage of the enzyme. Any two enoyl thioester reductases whi ch exhibit different stereospecificities are, therefore, predicted to have different pedigrees and unrelated amino acid sequences. An evalua tion of all the enoyl thioester reductases where both the stereochemic al course of reduction and the predicted amino acid sequence are known is shown to be entirely consistent with these predictions.