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 enzymes participates in a proton delivery system for binding and cleavage of dioxygen molecules. 6-Deoxyerythronolide B hydroxylase (P450eryF) is unusual in that the conserved threonine residue is replaced by alanine in this enzyme. On the basis of the 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, respectively. This hydrogen bonding network constitutes the proton delivery system whereby P450eryF maintains its catalytic activity in the absence of a threonine hydroxyl group in the conserved position. To further assess the role in the proton delivery system of hydroxyl groups around the active site, three mutant forms of P450eryF (A245S, S246A, and A245S/S246A) were constructed and characterized. In each case, decreased catalytic activity and increased uncoupling could be correlated with changes in the hydrogen bonding environment. These results suggest that Ser-246 does indeed indirectly participate in the proton shuttling pathway, and also strongly support our previous hypothesis that the C-5 hydroxyl group of the substrate participates in the acid-catalyzed dioxygen bond cleavage reaction.
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Department of Genetic Engineering, Institute of Agricultural Science and Technology, College of Agriculture, Chonnam National University, Kwangju, Republic of Korea.
Kim
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Han
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D001426:Bacterial Proteins; D002849:Chromatography, Gas; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004917:Erythromycin / Q000031:analogs & derivatives / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000737:chemistry; D006900:Hydroxylation; D007202:Indicators and Reagents; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D012694:Serine / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet
D001426:Bacterial Proteins; D007202:Indicators and Reagents; C034277:6-deoxyerythronolide B; D012694:Serine; D009249:NADP; D004917:Erythromycin; D003577:Cytochrome P-450 Enzyme System; D006861:Hydrogen Peroxide; D006899:Mixed Function Oxygenases; C054444:eryF protein, Saccharopolyspora erythraea