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-Deoxythronolide B hydroxylase (P450eryF) is unusual i n that the conserved threonine residue is replaced by alanine in this enzym e. on the basis Of the crystal structures of substrate-hound P450eryF it ha s been proposed that the C-5 hydroxyl group of the substrate and serine-246 of the enzyme form hydrogen bonds with water molecules S19 and 564. respec tively. This hydrogen bonding network constitutes the proton delivery. syst em whereby P450eryF maintains its catalytic activity in tl;e absence of a t hreonine hydroxyl group in the conserved position, To further assess the ro le in tile proton delivery system of hydroxyl groups around tile active sit e, three mutant forms of P450eryF (A245S, S246A, and A245S/S246A) were cons tructed and characterized, In each case, decreased catalytic activity and i ncreased uncoupling could be correlated with changes in the hydrogen bondin g environment, These results suggest bat Ser-246 does indeed indirectly par ticipate in the proton shuttling pathway, and also strongly support our pre vious hypothesis that the C-5 hydroxyl group of the substrate participates in the acid-catalyzed dioxygen bond cleavage reaction.