The role of serine-246 in cytochrome P450eryF-catalyzed hydroxylation of 6-deoxyerythronolide B

A strongly conserved threonine residue in the I-helix of cytochrome P450 en zymes participates in a proton delivery system for binding and cleavage of dioxygen molecules. 6-Deoxyerythronolide B hydroxylase (P450eryF) is unusua l in that the conserved threonine residue is replaced by alanine in this en zyme. On the basis of crystal structures of substrate-bound P450eryF it has been proposed that the C-5 hydroxyl group of the substrate and serine-246 of the enzyme form hydrogen bonds with water molecules 519 and 564, respect ively. This hydrogen bonding network constitutes the proton delivery system whereby P450eryF maintains its catalytic activity in the absence of a thre onine hydroxyl group in the conserved position. To further assess the role in the proton delivery system of hydroxyl groups around the active site, th ree mutant forms of P450eryF (A245S, S246A, and A245S/S246A) were construct ed and characterized. In each case, decreased catalytic activity and increa sed uncoupling could be correlated with changes in the hydrogen bonding env ironment. These results suggest that Ser-246 does indeed participate in the proton shuttling pathway, and also support our previous hypothesis that th e C-5 hydroxyl group of the substrate participates in the acid-catalyzed di oxygen bond cleavage reaction. (C) 2000 Academic Press.