Atomic Structure of Mycobacterium tuberculosis CYP121 to 1.06 Å Reveals Novel Features of Cytochrome P450

Leys, D.; Mowat, Christopher G.; McLean, Kirsty J.; Richmond, Alison; Chapman, Stephen K; Walkinshaw, Malcolm D.; Munro, Andrew W.

doi:10.1074/jbc.m209928200

Cited by 134 publications

(167 citation statements)

References 38 publications

(30 reference statements)

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“…Its aminopyrazole makes no clear interactions with the protein and points into the exterior water network of the substrate entry cavity. This is the same pyrazole binding mode observed previously for iodopyrazole binding to CYP121 (Figure 2 c, inset) 6. The iodine atom binds in the same orientation as the phenol ring of 6 , buried between the hydrophobic side chains of Val78, Phe168, Thr229, and Ala233.…”

supporting

confidence: 85%

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Overcoming the Limitations of Fragment Merging: Rescuing a Strained Merged Fragment Series Targeting Mycobacterium tuberculosis CYP121

et al. 2013

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Freedom to merge: A combination of crystal structure examination and in silico predictions made it possible to overcome the conformational limitations of fragment merging and escape the internal strain in a series of weakly binding merged fragments that target M. tuberculosis CYP121. The insights attained provide a new perspective and guide for prioritizing synthetic efforts toward fragment merging in future and ongoing fragment‐based ligand discovery campaigns.Permissions: WILEY-VCH

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supporting

confidence: 85%

“…The view and colors are the same as those described for panel a). The inset in panel c) shows an overlay of the comparative CYP121 binding positions of 6 and iodopyrazole from the 1.8 Å co‐crystal structure by Leys et al 6. (PDB ID: 1N4G).…”

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confidence: 99%

Overcoming the Limitations of Fragment Merging: Rescuing a Strained Merged Fragment Series Targeting Mycobacterium tuberculosis CYP121

et al. 2013

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“…The heme itself appears to exist in two distinct conformations as previously observed in the atomic resolution structure of CYP121 from M. tuberculosis (31), evidenced by persistent electron density in the difference map adjacent to the methyl groups of the pyrrole rings bearing the vinyl groups, when refined in either of two possible orientations. On the distal face of the heme iron, occupying the sixth coordination position, clear density was observed in the difference map for the ligand imidazole, which had presumably persisted from the nickel affinity column buffers used during purification of XplA-heme (Fig.…”

Section: Resultsmentioning

confidence: 63%

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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“…1 and 2). The P450 enzyme heme iron is ligated to the protein by a conserved cysteinate (the proximal ligand), with a water molecule usually present as the distal axial ligand (3,4). The P450 enzymes are found throughout nature and typically catalyze the reductive scission of dioxygen bound to the heme iron, frequently resulting in hydroxylation of an organic substrate.…”

mentioning

confidence: 99%

Flavocytochrome P450 BM3 Mutant A264E Undergoes Substrate-dependent Formation of a Novel Heme Iron Ligand Set

Girvan

Marshall

Lawson

et al. 2004

Journal of Biological Chemistry

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A conserved glutamate covalently attaches the heme to the protein backbone of eukaryotic CYP4 P450 enzymes. In the related Bacillus megaterium P450 BM3, the corresponding residue is Ala 264 . The A264E mutant was generated and characterized by kinetic and spectroscopic methods. A264E has an altered absorption spectrum compared with the wild-type enzyme (Soret maximum at ϳ420.5 nm). Fatty acid substrates produced an inhibitor-like spectral change, with the Soret band shifting to 426 nm. Optical titrations with long-chain fatty acids indicated higher affinity for A264E over the wild-type enzyme. The heme iron midpoint reduction potential in substrate-free A264E is more positive than that in wild-type P450 BM3 and was not changed upon substrate binding. EPR, resonance Raman, and magnetic CD spectroscopies indicated that A264E remains in the low-spin state upon substrate binding, unlike wild-type P450 BM3. EPR spectroscopy showed two major species in substrate-free A264E. The first has normal Cys-aqua iron ligation. The second resembles formateligated P450cam. Saturation with fatty acid increased the population of the latter species, suggesting that substrate forces on the glutamate to promote a Cys-Glu ligand set, present in lower amounts in the substratefree enzyme. A novel charge-transfer transition in the near-infrared magnetic CD spectrum provides a spectroscopic signature characteristic of the new A264E heme iron ligation state. A264E retains oxygenase activity, despite glutamate coordination of the iron, indicating that structural rearrangements occur following heme iron reduction to allow dioxygen binding. Glutamate coordination of the heme iron is confirmed by structural studies of the A264E mutant (Joyce, M. G.,

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Atomic Structure of Mycobacterium tuberculosis CYP121 to 1.06 Å Reveals Novel Features of Cytochrome P450

Cited by 134 publications

References 38 publications

Overcoming the Limitations of Fragment Merging: Rescuing a Strained Merged Fragment Series Targeting Mycobacterium tuberculosis CYP121

Overcoming the Limitations of Fragment Merging: Rescuing a Strained Merged Fragment Series Targeting Mycobacterium tuberculosis CYP121

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

Flavocytochrome P450 BM3 Mutant A264E Undergoes Substrate-dependent Formation of a Novel Heme Iron Ligand Set

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