Substrate Oxidation by Cytochrome P450 Enzymes

Montellano, Paul R. Ortiz de

doi:10.1007/0-387-27447-2_6

Cited by 237 publications

(292 citation statements)

References 328 publications

Supporting

Mentioning

279

Contrasting

Unclassified

Order By: Relevance

“…Previous in vivo experiments suggested that the protein encoded by ALK8 could hydroxylate fatty acids [14]. The well-known P450 enzymes that catalyze fatty acid hydroxylation are the members of the mammalian P450 4A family and P450 BM3 (CYP102) [1]. Amino acid sequence alignment of CYP52A21 with P450 4A1 and 4A3 revealed a high sequence similarity with a score of more than 70%, while the alignment with P450 BM3 was only 30% (Fig.…”

Section: Sequence Alignment With Other P450smentioning

confidence: 88%

“…The cytochrome P450 monooxygenases (CYPs or P450s) are members of a superfamily of heme-containing enzymes involved in the oxidative metabolism of endo-and xenobiotic chemicals [1]. These P450 enzymes are found throughout nature, from archaebacteria to humans [2].…”

Section: Introductionmentioning

confidence: 99%

“…Fatty acids are commonly hydroxylated by the mammalian CYP4 enzymes, a family of proteins that are characterized by their unique ability to hydroxylate the thermodynamically disfavored terminal methyl group of fatty acids rather than the default, thermodynamically favored internal chain carbons that are hydroxylated by other P450 enzymes [1]. Clearly, the CYP4 enzymes have evolved a strategy that allows them to specifically direct the reaction to the terminal (ω-) carbon of the fatty acid chain.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Functional expression and characterization of cytochrome P450 52A21 from Candida albicans

Kim

Cryle

Voss

et al. 2007

Archives of Biochemistry and Biophysics

Self Cite

View full text Add to dashboard Cite

Candida albicans contains ten putative cytochrome P450 (CYP) genes coding for enzymes that appear to play important roles in fungal survival and virulence. Here, we report the characterization of CYP52A21, a putative alkane/fatty acid hydroxylase. The recombinant CYP52A21 protein containing a 6 × (His)-tag was expressed in Escherichia coli and was purified. The purified protein, reconstituted with rat NADPH-cytochrome P450 reductase, ω-hydroxylated dodecanoic acid to give 12-hydroxydodecanoic acid, but to a lesser extent also catalyzed (ω-1)-hydroxylation to give 11-hydroxydodecanoic acid. When 12,12,12-d 3 -dodecanoic acid was used as the substrate, there was a major shift in the oxidation from the ω-to the (ω-1)-hydroxylated product. The regioselectivity of fatty acid hydroxylation was examined with the 12-iodo-, 12-bromo-, and 12-chlorododecanoic acids. Although all three 12-halododecanoic acids bound to CYP52A21 with similar affinities, the production of 12-oxododecanoic acid decreased as the size of the terminal halide increased. The regioselectivity of CYP52A21 fatty acid oxidation is thus consistent with presentation of the terminal end of the fatty acid chain for oxidation via a narrow channel that limits access to other atoms of the fatty acid chain. This constricted access, in contrast to that proposed for the CYP4A family of enzymes, does not involve covalent binding of the heme to the protein.

show abstract

Section: Sequence Alignment With Other P450smentioning

confidence: 88%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Functional expression and characterization of cytochrome P450 52A21 from Candida albicans

Kim

Cryle

Voss

et al. 2007

Archives of Biochemistry and Biophysics

Self Cite

View full text Add to dashboard Cite

show abstract

“…1 The P450-catalyzed reaction has been thought for three decades to be mediated by a Compound I-like ferryl species that is generated by reductive activation of molecular oxygen at the prosthetic heme iron center. Despite support by a variety of data for a Compund I-like ferryl species in the P450 catalytic cycle, it has remained elusive towards spectroscopic detection.…”

Section: Introductionmentioning

confidence: 99%

Selenolate Complexes of CYP101 and the Heme-Bound hHO-1/H25A Proximal Cavity Mutant

Jiang

Montellano

2008

Inorg. Chem.

Self Cite

View full text Add to dashboard Cite

Thiolate and selenolate complexes of CYP101 (P450cam) and the H25A proximal cavity mutant of heme-bound human heme oxygenase-1 (hHO-1) have been examined by UV-visible spectroscopy. Both thiolate and selenolate ligands bound to the heme distal side in CYP101 and gave rise to characteristic hyperporphyrin spectra. Thiolate ligands also bound to the proximal side of the heme in the cavity created by the H25A mutation in hHO-1, giving a Soret absorption similar to that of the H25C hHO-1 mutant. Selenolate ligands also bound to this cavity mutant under anaerobic conditions, but reduced the heme iron to the ferrous state as shown by formation of a ferrous-CO complex. Under aerobic conditions, the selenolate but not thiolate ligand was rapidly oxidized. These results indicate that selenocysteine-coordinated heme proteins will not be stable species in the absence of a redox potential stabilizing effect.Cytochrome P450 enzymes are a superfamily of heme proteins that employ a cysteine thiolate as the proximal ligand to the heme. 1 The P450-catalyzed reaction has been thought for three decades to be mediated by a Compound I-like ferryl species that is generated by reductive activation of molecular oxygen at the prosthetic heme iron center. Despite support by a variety of data for a Compund I-like ferryl species in the P450 catalytic cycle, it has remained elusive towards spectroscopic detection. 2,3 Conflicting results have also been obtained from determinations of the lifetime of the ferryl species using radical clock substrates. 4 Direct detection and characterization of the reactive oxidizing species in P450 enzymes would therefore be highly desirable. Recent computational studies have suggested that a seleno-CYP101 mutant in which the cysteine ligand is replaced by selenocysteine might provide an approach to investigation of the elusive P450 oxidizing species. 5 The Se-Compound I intermediate was predicted to form faster than the wild-type S-Compound I, and to be consumed more slowly. The increased lifetime of the intermediate suggested by the calculations indicated that it might become spectroscopically detectable.Targeted insertion of selenocysteine into a protein sequence through genetic code manipulations is a complex cotranslational process not easily adapted to P450 enzymes. 6,7 An alternative and straightforward approach to investigate the effect of ligands in heme proteins has been to use a cavity mutant in which the proximal amino acid ligand is replaced by a small and noncoordinating amino acid such as an alanine or glycine, creating a pocket that can accommodate exogenous unnatural ligands. A successful example of this is provided by the sperm whale myoglobin (Mb) H93G mutant, which has been extensively used for this purpose. 8,9 The crystal structures of β-mercaptoethanol and acetate-bound Mb H93G have recently confirmed that the exogenous ligands bind in the proximal ligation site.

show abstract

“…Non-productive H 2 O 2 generation was negligible with the T252N and T252N/V253T mutants, but, as previously observed, was dominant in the T252A mutant. Our results, and a structure model based on the crystal structures of the ferrous dioxygen complexes of P450 cam and its T252A mutant, suggest that Asn252 can stabilize the ferric hydroperoxy intermediate, preventing premature release of H 2 O 2 and enabling addition of the second proton to the distal oxygen to generate the catalytic ferryl species.The cytochrome P450 (CYP or P450) family of enzymes is involved in oxidative metabolism of a wide range of endo-and exogenous chemicals [1]. P450 cam (CYP101) from Pseudomonas putida, the structurally and biochemically best characterized P450 enzyme [2], catalyzes the regio-and stereospecific hydroxylation of camphor to 5-exo-hydroxycamphor.…”

mentioning

confidence: 99%

Efficient catalytic turnover of cytochrome P450cam is supported by a T252N mutation

Kim

Heo

Montellano

2008

Archives of Biochemistry and Biophysics

View full text Add to dashboard Cite

A Thr (or Ser) residue on the I-helix is a highly conserved structural feature of cytochrome P450 enzymes. It is believed to be indispensable as a proton delivery shuttle in the oxygen activation process. Previous work showed that P450 cin (CYP176A1), which contains an Asn instead of the conserved Thr, is fully functional in the catalytic oxidation of cineole [Hawkes, D.B. et al. (2002) J. Biol. Chem. 277, 27725-27732]. To determine whether the substitution of Asn for Thr is specific or general, the conserved Thr252 in P450 cam (CYP101) was mutated to generate the T252N, T252N/ V253T, and T252A mutants. Steady-state kinetic analysis of the oxidation of camphor by these mutants indicated that the T252N and T252N/V253T mutants have comparable turnover numbers but higher K m values relative to the wild-type enzyme. Spectroscopic binding assays indicate that the higher K m values reflect a decrease in the camphor binding affinity. Non-productive H 2 O 2 generation was negligible with the T252N and T252N/V253T mutants, but, as previously observed, was dominant in the T252A mutant. Our results, and a structure model based on the crystal structures of the ferrous dioxygen complexes of P450 cam and its T252A mutant, suggest that Asn252 can stabilize the ferric hydroperoxy intermediate, preventing premature release of H 2 O 2 and enabling addition of the second proton to the distal oxygen to generate the catalytic ferryl species.The cytochrome P450 (CYP or P450) family of enzymes is involved in oxidative metabolism of a wide range of endo-and exogenous chemicals [1]. P450 cam (CYP101) from Pseudomonas putida, the structurally and biochemically best characterized P450 enzyme [2], catalyzes the regio-and stereospecific hydroxylation of camphor to 5-exo-hydroxycamphor. As first established for P450 cam , the general P450 catalytic cycle involves (a) substrate binding, (b) reduction of the iron from the ferric to the ferrous state, (c) Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.Abbreviations used: CYP (P450), cytochrome P450; PCR, polymerase chain reaction; ORF, open reading frames; Pd, putidaredoxin; PdR, putidaredoxin reductase; TB, Terrific Broth; GC-MS, gas chromatography-mass spectrometry. K d indicates a dissociation constant estimated by measurement of the UV-visible changes in the P450 heme spectrum. An acid-alcohol side-chain pair, typically an Asp and Thr, is thought in most P450 enzymes to be a requirement for productive dioxygen activation in P450 catalysis [3]. The crystal structures of ferrous camphor-and CO-bound P450 cam suggest that Thr252 is a key catal...

show abstract

Substrate Oxidation by Cytochrome P450 Enzymes

Cited by 237 publications

References 328 publications

Functional expression and characterization of cytochrome P450 52A21 from Candida albicans

Functional expression and characterization of cytochrome P450 52A21 from Candida albicans

Selenolate Complexes of CYP101 and the Heme-Bound hHO-1/H25A Proximal Cavity Mutant

Efficient catalytic turnover of cytochrome P450cam is supported by a T252N mutation

Contact Info

Product

Resources

About