Role of Active Site Water Molecules and Substrate Hydroxyl Groups in Oxygen Activation by Cytochrome P450 158A2

Abstract

cytochrome P450 (CYP) genes in the actinomycete Streptomyces coelicolor, ordered active site water molecules (WAT505, WAT600, and WAT640), and hydroxyl groups of the substrate flaviolin were proposed to participate in proton transfer and oxygen cleavage in this monooxygenase. To probe their roles in catalysis, we have studied the crystal structures of a substrate analogue (2-hydroxy-1,4-naphthoquinone) complex with ferric CYP158A2 (2.15 Å ) and the flaviolin ferrous dioxygen-bound CYP158A2 complex (1.8 Å ). Catalytic activity toward 2-hydroxy-1,4-naphthoquinone was ϳ70-fold less than with flaviolin. In the ferrous dioxygen-bound flaviolin complex, the three water molecules in the ferric flaviolin complex still occupy the same positions and form hydrogen bonds to the distal dioxygen atom. These findings suggest that CYP158A2 utilizes substrate hydroxyl groups to stabilize active site water and further assist in the iron-linked dioxygen activation. A continuous hydrogen-bonded water network connecting the active site to the protein surface (bulk solvent) not present in the other two ferrous dioxygen-bound P450 structures (CYP101A1/P450 cam and CYP107A1/P450 eryF ) is proposed to participate in the proton-delivery cascade, leading to dioxygen bond scission. This ferrous-dioxygen structure suggests two classes of P450s based on the pathway of proton transfer, one using the highly conserved threonine in the I-helix (CYP101A1) and the other requiring hydroxyl groups of the substrate molecules either directly transferring protons (CYP107A1) or stabilizing a water pathway for proton transfer (CYP158A2). Cytochrome P450 (P450 or CYP)2 enzymes are ubiquitous hemebased monooxygenases in nature and are involved in the biosynthesis of physiologically important compounds as well as in drug and other xenobiotic metabolism (1-3). Some microbial P450s are associated with polyketide biosynthetic gene clusters where they catalyze late-stage stereo-and regiospecific oxidations (4 -7). For example, in Streptomyces coelicolor A3 (2) CYP158A2 is located in a three-gene operon, sco1206 -sco1208, that also contains a type III polyketide synthase 1,3,6,8-tetrahydroxynaphthalene synthase (8, 9) and a novel quinone-forming monooxygenase (momA) (10). This type III PKS catalyzes the sequential coupling of five molecules of malonyl-CoA to form 1,3,6,8-tetrahydroxynaphthalene, which is oxidized to flaviolin by momA. CYP158A2 then catalyzes an unusual phenolic oxidative C-C coupling reaction, in which two or three molecules of flaviolin are polymerized to biflaviolin or triflaviolin (7), red-brown pigments, which may afford physical protection to this soil bacterium, possibly against the deleterious effects of UV light radiation (11). During the general P450 catalytic cycle (1), dioxygen binding, protonation, and splitting of the oxygen-oxygen bond with generation of a perferryl iron complex (FeO 3ϩ ) are critical steps for product formation. The most detailed understanding of the proton transfer systems in P450s is from the investigations with...

Probing water micro-solvation in proteins by water catalysed proton-transfer tautomerism

“…The focus of probing water in proteins is on the cytochrome P450 superfamily because recent advances have generated more convincing data indicating that water molecules have pivotal roles in the organism metabolic pathways in P450s [31][32][33] . We herein selected the structurally pending TXAS [34][35][36] , an endoplasmic reticulum membrane protein containing five tryptophans, as a prototype to demonstrate the feasibility of (2,7-aza)Trp in probing water environments.…”

Regio- and Stereospecificity of Filipin Hydroxylation Sites Revealed by Crystal Structures of Cytochrome P450 105P1 and 105D6 from Streptomyces avermitilis

“…The Asp/Glu-Thr pair is conserved in CYP105P1 (Asp-240 -Thr-241) and CYP105D6 (Glu-246 -Thr-247). In the case of P450eryF and CYP158A2, the Thr residue is replaced with Ala, but a hydroxyl group of their substrates substitutes for the Thr and helps to deliver the protons (47,48). In the active site of CYP105P1, no hydroxyl group of the filipin I or 1Ј-hydroxyfilipin I substrate is positioned near the heme iron.…”

Double electron–electron resonance shows cytochrome P450cam undergoes a conformational change in solution upon binding substrate

“…Structurally distinct substrate binding sites have been observed for bacterial (4,10,11), microsomal (12)(13)(14)(15), and mitochondrial P450s (16). In addition, several P450s have been observed to undergo significant conformational change in these structural elements upon substrate or inhibitor binding (17)(18)(19)(20)(21)(22). However, it remains unclear whether conformational change plays an important role in all P450s.…”