4lht | pdb_00004lht
Crystal Structure of P450cin Y81F mutant, crystallized in 3 mM 1,8-cineole
Structural highlights
FunctionCINA_CITBR Involved in the degradation of cineol (eucalyptol). Catalyzes the initial hydroxylation of cineole exclusively at the pro-R carbon to give the (S)-6beta-hydroxycineole. Cineole is the natural substrate of CinA. Publication Abstract from PubMedIn P450cin, Tyr81, Asp241, Asn242, two water molecules, and the substrate participate in a complex H-bonded network. The role of this H-bonded network in substrate binding and catalysis has been probed by crystallography, spectroscopy, kinetics, isothermal titration calorimetry (ITC), and molecular dynamics. For the Y81F mutant, the substrate binds about 20-fold more weakly and Vmax decreases by about 30% in comparison to WT. The enhanced susceptibility of the heme to H2O2-mediated destruction in Y81F suggests that this mutant favors the open, low-spin conformational state. Asn242 H-bonds directly with the substrate, and replacing this residue with Ala results in water taking the place of the missing Asn side chain. This mutant exhibits a 70% decrease in activity. Crystal structures and molecular dynamics simulations of substrate-bound complexes show that the solvent has more ready access to the active site, especially for the N242A mutant. This accounts for about a 64% uncoupling of electron transfer from substrate hydroxylation. These data indicate the importance of the interconnected water network on substrate binding and on the open/closed conformational equilibrium, which are both critically important for maintaining high-coupling efficiency. P450cin Active Site Water: Implications for Substrate Binding and Solvent Accessibility.,Madrona Y, Hollingsworth SA, Khan B, Poulos TL Biochemistry. 2013 Jul 18. PMID:23829586[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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