Delta(3,5),Delta(2,4)-dienoyl-CoA isomerase is a multifunctional isomerase- A structural and mechanistic study

Delta (3,5),Delta (2,4)-Dienoyl-CoA isomerase (DI), an auxiliary enzyme of unsaturated fatty acid beta -oxidation, was purified from rat mitochondria and peroxisomes and subjected to N-terminal sequencing to facilitate a mech anistic study of this enzyme, The mature mitochondrial DI from rat heart wa s lacking its 34 N-terminal amino acid residues that have the properties of a mitochondrial targeting sequence. The peroxisomal isomerase was identifi ed as a product of the same gene with a truncated and ragged N terminus. Ex pression of the cDNA coding for the mature mitochondrial DI in Escherichia coli yielded an enzyme preparation that was as active as the native DI, Bec ause the recombinant DI also exhibited Delta (3,5,7)Delta (2,4,6)-trienoyl- CoA isomerase (TI) activity, both isomerases reside on the same protein, Mu tations of any of the 3 acidic amino acid residues located at the active si te (Modis, Y,, Filppula, S, A, Novikov, D, H., Norledge, B,, Hiltunen, J, K ., and Wierenga, R, K, (1998) Structure 6, 957-970) caused activity losses. In contrast to only a 10-fold decrease in activity upon replacement of Asp (176) by Ala, substitutions of Asp(204) by Asn and of Glu(196) by Gin resul ted in 10(5)-fold lower activities. Such activity losses are consistent wit h the direct involvement of these latter two residues in the proposed proto n transfers at carbons 2 and 6 or 8 of the substrates, Probing of the wild- type and mutants forms of the enzyme with 2,5-octadienoyl-CoA as substrate revealed low Delta (2),Delta (3)- enoyl-CoA isomerase and Delta (5),Delta ( 4)-enoyl-CoA isomerase activities catalyzed by Glu(196) and Asp(204), respe ctively. Altogether, these data reveal that positional isomerizations of th e diene and triene are facilitated by simultaneous proton transfers involvi ng Glu(196) and Asp(204) whereas each residue alone can catalyze, albeit le ss efficiently, a monoene isomerization.