Specific and non-specific effects of potassium cations on substrate–protein interactions in cytochromes P450cam and P450lin

Deprez, Eric; Gill, E. W.; Helms, Volkhard; Wade, Rebecca C.; Hoa, Gaston Hui Bon

doi:10.1016/s0162-0134(02)00467-1

Cited by 16 publications

(37 citation statements)

References 57 publications

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“…This must clearly be absent in the mutant, but its loss is not reflected in a change in the affinity of the protein for cineole. This is in line with the result observed for the Y96F P450cam mutant in which a H-bond from the protein to the substrate is lost without noticeable change in affinity (28,29). Taken as a whole, these results suggest that the architecture of the active site and, in particular, the heme environment is maintained in the N242A mutant, a conclusion borne out by subsequent structural characterization to be described next.…”

Section: Resultssupporting

confidence: 88%

The Critical Role of Substrate-Protein Hydrogen Bonding in the Control of Regioselective Hydroxylation in P450cin

Meharenna

Slessor

Cavaignac

et al. 2008

Journal of Biological Chemistry

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Cytochrome P450cin (CYP176A1) is a bacterial P450 isolated from Citrobacter braakii that catalyzes the hydroxylation of cineole to (S)-6␤-hydroxycineole. This initiates the biodegradation of cineole, enabling C. braakii to live on cineole as its sole source of carbon and energy. P450cin lacks the almost universally conserved threonine residue believed to be involved in dioxygen activation and instead contains an asparagine at this position (Asn-242). To investigate the role of Asn-242 in P450cin catalysis, it was converted to alanine, and the resultant mutant was characterized. The characteristic CO-bound spectrum and spectrally determined K D for substrate binding were unchanged in the mutant. The x-ray crystal structures of the substrate-free and -bound N242A mutant were determined and show that the only significant change is in a reorientation of the substrate such that (R)-6␣-hydroxycineole should be a major product. Molecular dynamics simulations of both wild type and mutant are consistent with the change in regio-and stereoselectivity predicted from the crystal structure. The mutation has only a modest effect on enzyme activity and on the diversion of the NADPHreducing equivalent toward unproductive peroxide formation. Product profile analysis shows that (R)-6␣-hydroxycineole is the main product, which is consistent with the crystal structure. These results demonstrate that Asn-242 is not a functional replacement for the conserved threonine in other P450s but, rather, is critical in controlling regioselective substrate oxidation.

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Section: Resultssupporting

confidence: 88%

The Critical Role of Substrate-Protein Hydrogen Bonding in the Control of Regioselective Hydroxylation in P450cin

Meharenna

Slessor

Cavaignac

et al. 2008

Journal of Biological Chemistry

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“…In CYP-S-CO, the largest confirmed K + -induced perturbations are observed to 15 N resonances of Cys 85 and Glu 94, each of which are directly bonded to proposed ligand carbonyl groups. The carbonyl 13 C of Glu 84 shows the largest confirmed perturbation of any signal upon replacement of K + by Tl + (Δδ = 116 Hz). However, while the carbonyl 13 C resonance of Glu 94 could not be detected in the HNCO spectrum of Tl + -perturbed CYP-S-CO, it can be detected in 100 mM KCl, and so is strongly perturbed as well.…”

Section: Discussion Localization and Oxidation State Dependence Of K mentioning

confidence: 99%

“…The carbonyl 13 C of Glu 84 shows the largest confirmed perturbation of any signal upon replacement of K + by Tl + (Δδ = 116 Hz). However, while the carbonyl 13 C resonance of Glu 94 could not be detected in the HNCO spectrum of Tl + -perturbed CYP-S-CO, it can be detected in 100 mM KCl, and so is strongly perturbed as well. With absolute shifts of 20 and 15 Hz, respectively, the Gly 93 and Tyr 96 carbonyl 13 C resonances are also perturbed by Tl + replacement of K + , although not as strongly as those of Glu 84 and Glu 94.…”

Section: Discussion Localization and Oxidation State Dependence Of K mentioning

confidence: 99%

“…We have now identified nearly all of the amide 1 H, 15 N correlations in the vicinity of the proposed K + binding site in CYP-S-CO using multidimensional NMR methods, as well as Cα and CO 13 Residues 86 and 89 are both prolines and hence do not contribute correlations to NH-detected 2D and 3D NMR experiments, although Cα and Cβ correlations are available for these residues through the HNCA, HN(CO)CA and HNCACB data sets. Combined with previous assignments, we can comprehensively survey the effects of K + binding on secondary structural features in CYP101.…”

Section: Structural Perturbations In Cyp-s-co and Cyp-s Due To K + Bimentioning

confidence: 94%

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Specific Effects of Potassium Ion Binding on Wild-Type and L358P Cytochrome P450cam

et al. 2006

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The camphor monoxygenase cytochrome P450 cam (CYP101) requires potassium ion (K + ) to drive formation of the characteristic high-spin state of the heme Fe +3 upon substrate binding. Amide 1 H, 15 N correlations in perdeuterated [U-15 N] CYP101 were monitored as a function of K + concentration by 2D-TROSY-HSQC in both camphor-bound oxidized (CYP-S) and camphor-and CO-bound reduced CYP101 (CYP-S-CO). In both forms, K + -induced spectral perturbations are detected in the vicinity of the K + binding site proposed from crystallographic structures, but are larger and more widespread structurally in CYP-S than in CYP-S-CO. In CYP-S-CO, K + -induced perturbations occur primarily near the proposed K + binding site in the B-B' loop and B' helix, which are also perturbed by binding of effector, putidaredoxin (Pdx). The spectral effects of K + binding in CYP-S-CO oppose those observed upon Pdx r titration. However, Pdx r titration of CYP-S-CO in the absence of K + results in multiple conformations. The spin-state equilibrium in the L358P mutant of CYP101 is more sensitive to K + concentration than WT CYP101, consistent with a hypothesis that L358P preferentially populates conformations enforced by Pdx binding in WT CYP101. Thallium (I), a K + mimic, minimizes the effects of Pdx titration on the NMR spectrum of CYP-S-CO, but is competent to replace K + in driving the formation of high-spin CYP-S. These observations suggest that the role of K + is to stabilize conformers of CYP-S that drive the spin state change prior to the first electron transfer, and that K + stabilizes the CYP-S-CO conformer that interacts with Pdx. However, upon binding of Pdx, further conformational changes occur that disfavor K + binding.

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“…Previous studies investigating the effects of ionic strength on the CYP spin state have used subzero temperatures, various pHs, presence of cosolvents, presence of substrates, and elevated pressures (8,9,10,13,24,25,31,32,37,38), due to the relative insensitivities of the spin states of many CYPs to modulation by ionic strength in the absence of substrate. We are currently performing further investigations on the modulation of CYP164A2 spin state by ionic strength.…”

Section: Figmentioning

confidence: 99%

Identification, Characterization, and Azole-Binding Properties of Mycobacterium smegmatis CYP164A2, a Homolog of ML2088, the Sole Cytochrome P450 Gene of Mycobacterium leprae

Warrilow

Jackson

Parker

et al. 2009

Antimicrob Agents Chemother

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The genome sequence of Mycobacterium leprae revealed a single open reading frame, ML2088 (CYP164A1), encoding a putative full-length cytochrome P450 monooxygenase and 12 pseudogenes. We have identified a homolog of ML2088 in Mycobacterium smegmatis and report here the cloning, expression, purification, and azole-binding characteristics of this cytochrome P450 (CYP164A2). CYP164A2 is 1,245 bp long and encodes a protein of 414 amino acids and molecular mass of 45 kDa. CYP164A2 has 60% identity with Mycobacterium leprae CYP161A1 and 66 to 69% identity with eight other mycobacterial CYP164A1 homologs, with three identified highly conserved motifs. Recombinant CYP164A2 has the typical spectral characteristics of a cytochrome P450 monooxygenase, predominantly in the ferric low-spin state. Unusually, the spin state was readily modulated by increasing ionic strength at pH 7.5, with 50% high-spin occupancy achieved with 0.14 M NaCl. CYP164A2 bound clotrimazole, econazole, and miconazole strongly (K d , 1.2 to 2.5 M); however, strong binding with itraconazole, ketoconazole, and voriconazole was only observed in the presence of 0.5 M NaCl. Fluconazole did not bind to CYP164A2 at pH 7.5 and no discernible type II binding spectrum was observed.Leprosy has afflicted humanity through the ages and remains today a serious and disfiguring condition in communities throughout the developing world. Immunization (18) and multidrug treatment programs have been effective in reducing morbidity and mortality due to lepromatous leprosy, but the incidence of new infections, at 680,000 per annum (36), remains high. Genome sequencing projects have been completed for several mycobacterial species, including both Mycobacterium leprae, the etiologic agent of leprosy, and Mycobacterium tuberculosis. A common objective of these schemes is to identify potential targets for the development of novel antimycobacterial compounds. Unusually for bacteria, the actinomycetes such as mycobacteria and streptomycetes contain substantial numbers of genes encoding cytochromes P450 (CYPs) or P450 pseudogenes (6, 23). These enzymes carry out a wide range of monooxygenation reactions involved in biocatalysis, secondary metabolism, and detoxification. CYPs are ubiquitous throughout the eukaryotes but are relatively uncommon in prokaryotes, with most prokaryotes containing no CYP genes. The unexpected discovery in M. tuberculosis of an ortholog of CYP51 (3), the sterol 14␣-demethylase of eukaryotes (19), proved a major catalyst for investigations into the CYP complements (CYPomes) of mycobacteria, as CYP51 is the target for azole antifungal drugs that have been shown to possess antimycobacterial properties (1,5,15,35).Most azole antifungal compounds show selective targeting of CYP51, having an 8-to 128-fold-higher affinity for fungal CYP51s relative to the orthologous enzyme present in the host (29). These compounds act by inhibiting the fungal sterol biosynthesis pathway, which does not exist in mycobacteria. The abundance of P450 proteins in mycobacterial species...

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Specific and non-specific effects of potassium cations on substrate–protein interactions in cytochromes P450cam and P450lin

Cited by 16 publications

References 57 publications

The Critical Role of Substrate-Protein Hydrogen Bonding in the Control of Regioselective Hydroxylation in P450cin

The Critical Role of Substrate-Protein Hydrogen Bonding in the Control of Regioselective Hydroxylation in P450cin

Specific Effects of Potassium Ion Binding on Wild-Type and L358P Cytochrome P450cam

Identification, Characterization, and Azole-Binding Properties of Mycobacterium smegmatis CYP164A2, a Homolog of ML2088, the Sole Cytochrome P450 Gene of Mycobacterium leprae

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