Crystal Structures of the Ferrous Dioxygen Complex of Wild-type Cytochrome P450eryF and Its Mutants, A245S and A245T

Nagano, Shingo; Cupp‐Vickery, Jill R.; Poulos, T.L.

doi:10.1074/jbc.m501732200

Cited by 91 publications

(115 citation statements)

References 25 publications

(26 reference statements)

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“…4) and this, in conjunction with the kinetic data on the Ala-395 3 Thr mutant, may suggest a role therefore in substrate binding by that cavity. Equivalent suppression of activity in the hydroxylation of 6-DEB was observed for Ala-Thr mutants of P450-EryF in this position (40), and this was attributed to steric factors, because P450-EryF-Ala-Thr mutants were still able to hydroxylate the smaller substrate testosterone (41). A role for the Ala-395 to Val-391 water cavity in substrate binding, as opposed to heme-oxygen intermediate stabilization, would also seem reasonable given that XplA does not require oxygen as such, at least in the anaerobic pathway to RDX that culminates in the metabolite methylene dinitramine 6 (Fig.…”

Section: Structure Of Xpla-hemementioning

confidence: 94%

The 1.5-Å Structure of XplA-heme, an Unusual Cytochrome P450 Heme Domain That Catalyzes Reductive Biotransformation of Royal Demolition Explosive

Sabbadin

Jackson

Haider

et al. 2009

Journal of Biological Chemistry

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XplA is a cytochrome P450 of unique structural organization, consisting of a heme-domain that is C-terminally fused to its native flavodoxin redox partner. XplA, along with flavodoxin reductase XplB, has been shown to catalyze the breakdown of the nitramine explosive and pollutant hexahydro-1,3,5-trinitro-1,3,5-triazine (royal demolition explosive) by reductive denitration. The structure of the heme domain of XplA (XplA-heme) has been solved in two crystal forms: as a dimer in space group P2 1 to a resolution of 1.9 Å and as a monomer in space group P2 1 2 1 2 to a resolution of 1.5 Å , with the ligand imidazole bound at the heme iron. Although it shares the overall fold of cytochromes P450 of known structure, XplA-heme is unusual in that the kinked I-helix that traverses the distal face of the heme is broken by Met-394 and Ala-395 in place of the well conserved Asp/Glu plus Thr/Ser, important in oxidative P450s for the scission of the dioxygen bond prior to substrate oxygenation. The heme environment of XplA-heme is hydrophobic, featuring a cluster of three methionines above the heme, including Met-394. Imidazole was observed bound to the heme iron and is in close proximity to the side chain of Gln-438, which is situated over the distal face of the heme. Imidazole is also hydrogenbonded to a water molecule that sits in place of the threonine side-chain hydroxyl exemplified by Thr-252 in Cyt-P450cam. Both Gln-438 3 Ala and Ala-395 3 Thr mutants of XplA-heme displayed markedly reduced activity compared with the wild type for royal demolition explosive degradation when combined with surrogate electron donors. Royal demolition explosive (RDX)2 or cyclotrimethylenetrinitramine 1 (see Fig. 1) is a widely used explosive compound with both military and civil applications. The extensive global usage of RDX has resulted in concerns over environmental contamination, because it is both recalcitrant to degradation, leading to contamination in soil and ground water, and a potent convulsant and possible carcinogen. The bioremediation of RDX has thus been the focus of increasing research in recent years, with a number of bacterial strains reported to catalyze its degradation (1-3). Among these, Bruce and co-workers, using a selective enrichment technique, isolated Rhodococcus rhodochrous strain 11Y from an RDX-contaminated site, which was able to grow on RDX as the sole nitrogen source (4). The products of biotransformation of RDX by this bacterium were shown to be nitrite and formaldehyde. A gene cluster in strain 11Y essential for RDX degradation was identified and shown to contain a novel cytochrome P450, termed XplA, and a redox partner, XplB, which were shown together to be capable of catalyzing the biotransformation of RDX in vitro (5). Near identical xplA and xplB genes have now been identified in strains within the Actinomycetales isolated from geographically distinct sites (6). Interestingly, these genes have been found to be plasmid encoded providing compelling evidence for recent lateral gene transfer. In the intere...

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Section: Structure Of Xpla-hemementioning

confidence: 94%

The 1.5-Å Structure of XplA-heme, an Unusual Cytochrome P450 Heme Domain That Catalyzes Reductive Biotransformation of Royal Demolition Explosive

Sabbadin

Jackson

Haider

et al. 2009

Journal of Biological Chemistry

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“…Interestingly, in CYP158A2 the residue position of the conserved threonine in the I-helix is also occupied by Ala 245 as observed in the CYP107A1 (18). In CYP107A1 the postulated crucial catalytic water molecule (WAT519) present in the ferric enzyme is absent in the ferrous dioxygen complex (19). To explore the unique features of the CYP158A2 proton transfer system, we determined the catalytic activity of the sub-* This work was supported by National Institutes of Health Grants GM37942 (to M. R. W.), GM69970 (to M. R. W. and F. P. G.), ES00267 (to F. P. G. and M. R. W.).…”

mentioning

confidence: 92%

“…Preparation of the Ferrous Dioxygen-bound Complex-The procedure for preparation of the ferrous dioxygen-bound complex was described by Nagano et al (19). CYP158A2 ferric flaviolin-bound crystals were transferred into cryobuffer (50 mM bis-Tris (pH 6.5), 1 mM flaviolin, 25% polyethylene glycol 3350, and 20% glycerol, v/v), washed for 5 min, and then reduced with 1-5 mM sodium dithionite under anaerobic conditions (N 2 ) in a glove box (Fisher, Vernon Hills, IL).…”

Section: Methodsmentioning

confidence: 99%

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

Zhao

Guengerich

Voehler

et al. 2005

Journal of Biological Chemistry

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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...

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“…By metabolic engineering and in future synthetic biology they will be incorporated into strategies to produce, or enhance, known and new drugs sustainably using microbes [109,110]. Among bacterial CYPs probably the earliest antibiotic biosynthesis CYP gene and protein studied in detail was P450 eryF (CYP107A1) involved in C6-hydroxylation of the macrolide 6-deoxyerythronolide B during erythromycin biosynthesis in the bacterium Saccharopolyspora ertherea [131,132]. As mentioned above, many pathways of bacterial and fungal secondary metabolism contain CYPs involved in multi-step oxidation, rearrangements, epoxidations and heteroatom oxidation.…”

Section: Microbes Cytochromes P450 and Human Healthmentioning

confidence: 99%

Microbial cytochromes P450: biodiversity and biotechnology. Where do cytochromes P450 come from, what do they do and what can they do for us?

Kelly

2013

Phil. Trans. R. Soc. B

181

131

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The first eukaryote genome revealed three yeast cytochromes P450 (CYPs), hence the subsequent realization that some microbial fungal genomes encode these proteins in 1 per cent or more of all genes (greater than 100) has been surprising. They are unique biocatalysts undertaking a wide array of stereo-and regio-specific reactions and so hold promise in many applications. Based on ancestral activities that included 14a-demethylation during sterol biosynthesis, it is now seen that CYPs are part of the genes and metabolism of most eukaryotes. In contrast, Archaea and Eubacteria often do not contain CYPs, while those that do are frequently interesting as producers of natural products undertaking their oxidative tailoring. Apart from roles in primary and secondary metabolism, microbial CYPs are actual/potential targets of drugs/agrochemicals and CYP51 in sterol biosynthesis is exhibiting evolution to resistance in the clinic and the field. Other CYP applications include the first industrial biotransformation for corticosteroid production in the 1950s, the diversion into penicillin synthesis in early mutations in fungal strain improvement and bioremediation using bacteria and fungi. The vast untapped resource of orphan CYPs in numerous genomes is being probed and new methods for discovering function and for discovering desired activities are being investigated.

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Crystal Structures of the Ferrous Dioxygen Complex of Wild-type Cytochrome P450eryF and Its Mutants, A245S and A245T

Cited by 91 publications

References 25 publications

The 1.5-Å Structure of XplA-heme, an Unusual Cytochrome P450 Heme Domain That Catalyzes Reductive Biotransformation of Royal Demolition Explosive

The 1.5-Å Structure of XplA-heme, an Unusual Cytochrome P450 Heme Domain That Catalyzes Reductive Biotransformation of Royal Demolition Explosive

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

Microbial cytochromes P450: biodiversity and biotechnology. Where do cytochromes P450 come from, what do they do and what can they do for us?

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