Catalytic mechanism of scytalone dehydratase from Magnaporthe grisea

The catalytic mechanism of scytalone dehydratase was examined by studying alternative substrates and site-directed mutations of active-site residues. Searches for an eno1 intermediate by looking for a half-reaction with authentic scytalone and 3,4-dihydro-6,8-dihydroxy-1-(2H)- 2[¹³C]naphthalenone were negative. An alternative substrate, 2,3-dihydro- 2,5-dihydroxy-4H-benzopyran-4-one (DDBO), was nearly equal to scytalone as substrate for the enzyme, 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.