Oxygen activation and redox partner binding in cytochromes P450

2013 ◽  
Vol 60 (1) ◽  
pp. 128-133 ◽  
Author(s):  
Thomas L. Poulos ◽  
Yarrow Madrona
Keyword(s):  
Cytochromes P450 ◽  
Oxygen Activation ◽  
Redox Partner

scholarly journals A Novel Thermostable Cytochrome P450 from Sequence-Based Metagenomics of Binh Chau Hot Spring as a Promising Catalyst for Testosterone Conversion

Catalysts ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1083 ◽  
Author(s):  
Kim-Thoa Nguyen ◽  
Ngọc-Lan Nguyen ◽  
Nguyen Van Tung ◽  
Huy Hoang Nguyen ◽  
Mohammed Milhim ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Retinoic Acid ◽  
Amino Acid Sequence Identity ◽  
Hot Spring ◽  
Cytochromes P450 ◽  
Reading Frame ◽  
Redox Partner ◽  
Redox Partners ◽  
Identification And Characterization ◽  
Low Activity

Biotechnological applications of cytochromes P450 show difficulties, such as low activity, thermal and/or solvent instability, narrow substrate specificity and redox partner dependence. In an attempt to overcome these limitations, an exploitation of novel thermophilic P450 enzymes from nature via uncultured approaches is desirable due to their great advantages that can resolve nearly all mentioned impediments. From the metagenomics library of the Binh Chau hot spring, an open reading frame (ORF) encoding a thermostable cytochrome P450—designated as P450-T3—which shared 66.6% amino acid sequence identity with CYP109C2 of Sorangium cellulosum So ce56 was selected for further identification and characterization. The ORF was synthesized artificially and heterologously expressed in Escherichia coli C43(DE3) using the pET17b system. The purified enzyme had a molecular weight of approximately 43 kDa. The melting temperature of the purified enzyme was 76.2 °C and its apparent half-life at 60 °C was 38.7 min. Redox partner screening revealed that P450-T3 was reduced well by the mammalian AdR-Adx4-108 and the yeast Arh1-Etp1 redox partners. Lauric acid, palmitic acid, embelin, retinoic acid (all-trans) and retinoic acid (13-cis) demonstrated binding to P450-T3. Interestingly, P450-T3 also bound and converted testosterone. Overall, P450-T3 might become a good candidate for biocatalytic applications on a larger scale.

MECHANISTIC ENZYMOLOGY OF OXYGEN ACTIVATION BY THE CYTOCHROMES P450

2002 ◽  
Vol 34 (4) ◽  
pp. 691-708 ◽  
Author(s):  
Thomas M. Makris ◽  
Roman Davydov ◽  
Ilia G. Denisov ◽  
Brian M. Hoffman ◽  
Stephen G. Sligar
Keyword(s):  
Cytochromes P450 ◽  
Oxygen Activation

scholarly journals Structure and function of the cytochrome P450 peroxygenase enzymes

2018 ◽  
Vol 46 (1) ◽  
pp. 183-196 ◽  
Author(s):  
Andrew W. Munro ◽  
Kirsty J. McLean ◽  
Job L. Grant ◽  
Thomas M. Makris
Keyword(s):  
Fatty Acids ◽  
Cytochromes P450 ◽  
Structural Data ◽  
Short Review ◽  
Biochemical Properties ◽  
Organic Substrates ◽  
Reactive Iron ◽  
Redox Partner ◽  
Wide Range ◽  
And Function

The cytochromes P450 (P450s or CYPs) constitute a large heme enzyme superfamily, members of which catalyze the oxidative transformation of a wide range of organic substrates, and whose functions are crucial to xenobiotic metabolism and steroid transformation in humans and other organisms. The P450 peroxygenases are a subgroup of the P450s that have evolved in microbes to catalyze the oxidative metabolism of fatty acids, using hydrogen peroxide as an oxidant rather than NAD(P)H-driven redox partner systems typical of the vast majority of other characterized P450 enzymes. Early members of the peroxygenase (CYP152) family were shown to catalyze hydroxylation at the α and β carbons of medium-to-long-chain fatty acids. However, more recent studies on other CYP152 family P450s revealed the ability to oxidatively decarboxylate fatty acids, generating terminal alkenes with potential applications as drop-in biofuels. Other research has revealed their capacity to decarboxylate and to desaturate hydroxylated fatty acids to form novel products. Structural data have revealed a common active site motif for the binding of the substrate carboxylate group in the peroxygenases, and mechanistic and transient kinetic analyses have demonstrated the formation of reactive iron-oxo species (compounds I and II) that are ultimately responsible for hydroxylation and decarboxylation of fatty acids, respectively. This short review will focus on the biochemical properties of the P450 peroxygenases and on their biotechnological applications with respect to production of volatile alkenes as biofuels, as well as other fine chemicals.

scholarly journals Conformational selectivity in cytochrome P450 redox partner interactions

2016 ◽  
Vol 113 (31) ◽  
pp. 8723-8728 ◽  
Author(s):  
Scott A. Hollingsworth ◽  
Dipanwita Batabyal ◽  
Brian D. Nguyen ◽  
Thomas L. Poulos
Keyword(s):  
Cytochrome P450 ◽  
Structural Changes ◽  
Cytochromes P450 ◽  
Substrate Oxidation ◽  
Heme Iron ◽  
Spectral Studies ◽  
Titration Calorimetry ◽  
Nmr Studies ◽  
Redox Partner ◽  
Open Conformation

The heme iron of cytochromes P450 must be reduced to bind and activate molecular oxygen for substrate oxidation. Reducing equivalents are derived from a redox partner, which requires the formation of a protein–protein complex. A subject of increasing discussion is the role that redox partner binding plays, if any, in favoring significant structural changes in the P450s that are required for activity. Many P450s now have been shown to experience large open and closed motions. Several structural and spectral studies indicate that the well-studied P450cam adopts the open conformation when its redox partner, putidaredoxin (Pdx), binds, whereas recent NMR studies indicate that this view is incorrect. Given the relevance of this discrepancy to P450 chemistry, it is important to determine whether Pdx favors the open or closed form of P450cam. Here, we have used both computational and experimental isothermal titration calorimetry studies that unequivocally show Pdx favors binding to the open form of P450cam. Analyses of molecular-dynamic trajectories also provide insights into intermediate conformational states that could be relevant to catalysis.

Proton Relay Network in P450cam is Formed Upon Docking of its Redox Partner Putidaredoxin (Pdx)

2019 ◽  
Author(s):  
Ilke Ugur Marion ◽  
Prasanna Chandrasekhar
Keyword(s):  
Secondary Structure ◽  
Cytochromes P450 ◽  
Direct Interaction ◽  
Relay Networks ◽  
Relay Network ◽  
Wild Type ◽  
Redox Partner ◽  
Proton Relay ◽  
Cleavage Step ◽  
First Time

Cytochromes P450 are versatile heme-based enzymes responsible for vital life processes. Of these, P450cam (substrate camphor) has been most studied. Despite this, precise mechanisms of the key O-O cleavage step remain elusive to date; effects observed in various enzyme mutants remain unexplained. We have carried out extended (up to 1000 ns) MM-MD and follow-on QM/MM computations, both on the well-studied FeOO state and, for the first time, on Cpd(0). Our simulations include (all camphor-bound) : (1) WT (wild type), FeOO state. (2) WT, Cpd(0). (3) Pdx-docked-WT, FeOO state. (4) Pdx-docked WT, Cpd(0). (5) Pdx-docked T252A mutant, Cpd(0). Among our key findings, for the first time to our knowledge: (1) Effect of Pdx docking goes far beyond that indicated in prior studies: it leads to specific alterations in secondary structure that create the crucial proton relay network. (2) The specific proton relay networks we identify are FeOO(H)---T252---nH2O---D251 in WT and FeOO(H)---nH2O---D251 in T252A mutant; both occur with Pdx docking. (3) Direct interaction of D251 with -FeOOH is, respectively, rare/frequent in WT/T252A mutant. (4) T252 is in the proton relay network. (5) Positioning of camphor is crucial: when camphor is part of H-bonding network, coupling is facilitated.<br>

scholarly journals Regulation of Microsomal P450, Redox Partner Proteins, and Steroidogenesis in the Developing Testes of the Neonatal Pig

Endocrinology ◽  
2002 ◽  
Vol 143 (9) ◽  
pp. 3361-3369 ◽  
Author(s):  
F. M. Moran ◽  
J. J. Ford ◽  
C. J. Corbin ◽  
S. M. Mapes ◽  
V. C. Njar ◽  
...  
Keyword(s):  
Gnrh Antagonist ◽  
Cytochromes P450 ◽  
Cytochrome B5 ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Reduced Form ◽  
Testicular Development ◽  
Aromatase Activity ◽  
P450 Reductase ◽  
Redox Partner ◽  
Partner Proteins

Abstract Testicular growth and plasma androgen concentrations increase markedly in the first weeks of neonatal life of pigs. The regulation of steroidogenesis through this period was examined by measuring total microsomal cytochromes P450 (P450), 17α-hydroxylase/17,20-lyase P450 (P450c17) and aromatase P450 (P450arom) enzyme activities, and the redox partner proteins nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-cytochrome P450 reductase (reductase) and cytochrome b5 in testicular microsomes. Testes were collected from 1–24 d of age, and testicular development was suppressed by a GnRH antagonist in some animals from d 1–14. Both 17/20-lyase and aromatase activities increased from d 1–7 but not thereafter, and 17–20-lyase activity was always at least 200-fold higher than aromatase activity. Reductase decreased in wk 1, then increased to d 24. No changes were seen in cytochrome b5 expression. GnRH antagonist treatment suppressed plasma LH, testosterone and testes growth to d 14. 17,20-Lyase and aromatase activities in testicular microsomes were reduced by 20% and 50%, respectively. Total microsomal P450 concentration was reduced by 50% on d 7, but there was no effect of treatment on reductase or cytochrome b5 expression. These data support the hypothesis that the rise in neonatal testicular androgen secretion is more likely due to gonadotropin-stimulated gonadal growth, rather than specific P450c17 expression. Neither P450c17 nor P450arom can account for the decline in total microsomal P450. Reductase and cytochrome b5 expression appears to be constitutive, but reductase levels saturate both P450c17 and P450arom.

scholarly journals Steroidogenesis during postnatal testicular development of Galea spixii

Reproduction ◽  
2017 ◽  
Vol 154 (5) ◽  
pp. 645-652
Author(s):  
P R S Santos ◽  
F D Oliveira ◽  
M A M Arroyo ◽  
M F Oliveira ◽  
P Castelucci ◽  
...  
Keyword(s):  
Sexual Development ◽  
Cytochromes P450 ◽  
Quantitative Polymerase Chain Reaction ◽  
Testicular Development ◽  
Androgen Synthesis ◽  
Redox Partner ◽  
Estrogen Synthesis ◽  
Gene Transcripts ◽  
Pubertal Stages ◽  
Polymerase Chain

The androgen/estrogen balance is essential for normal sexual development and reproduction in mammals. Studies performed herein investigated the potential for estrogen synthesis in cells of the testes of a hystricomorph rodent, Galea spixii. The study characterized the expression of the key enzymes responsible for estrogen and androgen synthesis, cytochromes P450 aromatase (P450arom), 17α-hydroxylase/17,20-lyase (P450c17) respectively, as well as the redox partner NADPH cytochrome P450 oxido-reductase (CPR) required to support electron transfer and catalysis of these P450s, by immunohistochemistry (IHC) and quantitative polymerase chain reaction (qPCR) analysis, throughout postnatal sexual development. Testes (immature, pre-pubertal, pubertal and post-pubertal) were collected, fixed for IHC (CYP19, CYP17 and CPR) and stored frozen for qPCR for the relevant gene transcripts (Cyp19a1 and Cyp17a1). Expression of P450c17 was significantly elevated at the pre-pubertal and pubertal stages. Based on IHC, P450c17 was expressed only in Leydig cell clusters. The expression of P450arom was detectable at all stages of sexual development of Galea spixii. IHC data suggest that estrogen synthesis was not restricted to somatic cells (Leydig cells/Sertoli cells), but that germ cells may also be capable of converting androgens into estrogens, important for testicular function and spermatogenesis.

Optimization of redox partner interactions with cytochromes P450 (CYP)

2016 ◽  
Vol 33 ◽  
pp. S102
Author(s):  
Tanja Sagadin ◽  
Jan Riehm ◽  
Michael Hutter ◽  
Rita Bernhardt
Keyword(s):  
Cytochromes P450 ◽  
Redox Partner

scholarly journals Evaluation of P450 monooxygenase activity in lyophilized recombinant E. coli cells compared to resting cells

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Thomas Hilberath ◽  
Alessandra Raffaele ◽  
Leonie M. Windeln ◽  
Vlada B. Urlacher
Keyword(s):  
Cytochromes P450 ◽  
Rhodococcus Erythropolis ◽  
Resting Cells ◽  
P450 Monooxygenase ◽  
Monooxygenase Activity ◽  
Whole Cells ◽  
E Coli ◽  
Redox Partner ◽  
Starting Point ◽  
P450 Activity

AbstractCytochromes P450 catalyze oxidation of chemically diverse compounds and thus offer great potential for biocatalysis. Due to the complexity of these enzymes, their dependency of nicotinamide cofactors and redox partner proteins, recombinant microbial whole cells appear most appropriate for effective P450-mediated biocatalysis. However, some drawbacks exist that require individual solutions also when P450 whole-cell catalysts are used. Herein, we compared wet resting cells and lyophilized cells of recombinant E. coli regarding P450-catalyzed oxidation and found out that lyophilized cells are well-appropriate as P450-biocatalysts. E. coli harboring CYP105D from Streptomyces platensis DSM 40041 was used as model enzyme and testosterone as model substrate. Conversion was first enhanced by optimized handling of resting cells. Co-expression of the alcohol dehydrogenase from Rhodococcus erythropolis for cofactor regeneration did not affect P450 activity of wet resting cells (46% conversion) but was crucial to obtain sufficient P450 activity with lyophilized cells reaching a conversion of 72% under the same conditions. The use of recombinant lyophilized E. coli cells for P450 mediated oxidations is a promising starting point towards broader application of these enzymes.

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