Catalytic mechanism of scytalone dehydratase from Magnaporthe grisea

The catalytic mechanism of scytalone dehydratase was examined by studying a lternative substrates and site-directed mutations of active-site residues. Searches for an enol intermediate by looking for a half-reaction with authe ntic scytalone and 3,4-dihydro-6,8-dihydroxy-1-(2H)-2-[C-13]naphthalenone w ere negative. An alternative substrate, 2,3-dihydro-2,5-dihydroxy-4H-benzop yrstn-4-one (DDBO), was nearly equal to scytalone as substrate for the enzy me, and DDBO's anomeric effect in stabilizing a partial carbocation center at C3 does not substantially contribute to the mechanism. Kinetic analysis of site-directed mutations of active-site amino acid side chains within the enzyme's active site provided an account for the role of these residues in the enzyme-catalyzed dehydration reactions. A concerted E2 elimination for the catalytic mechanism is proposed. (C) 1999 Society of Chemical Industry .