Heterologous expression and functional characterization of the NADPH-cytochrome P450 reductase from Capsicum annuum

Lee, Ga-Young; Kim, Hyun Min; Hoon, Sang; Park, Se Hee; Joung, Young Hee; Yun, Chul‐Ho

doi:10.1016/j.plaphy.2014.05.010

Cited by 20 publications

(21 citation statements)

References 27 publications

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“…As the electrons donors, CPRs transfer two electrons from NADPH through the FAD and FMN cofactors into the central heme iron of P450s [ 22 ]. CPRs were proven to be presented in most living organisms, for instance, yeasts, plants and animals [ 23 – 30 ]. These CPRs were functionally characterized to donate electrons to P450s to support the oxidation reactions catalyzed by P450s [ 23 – 30 ].…”

Section: Introductionmentioning

confidence: 99%

“…CPRs were proven to be presented in most living organisms, for instance, yeasts, plants and animals [ 23 – 30 ]. These CPRs were functionally characterized to donate electrons to P450s to support the oxidation reactions catalyzed by P450s [ 23 – 30 ]. Herein we report the molecular cloning, heterologous overexpression, and functional characterization of CamCPR , an NADPH-dependent CPR encoding gene from C .…”

Section: Introductionmentioning

confidence: 99%

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Molecular Cloning, Heterologous Expression, and Functional Characterization of an NADPH-Cytochrome P450 Reductase Gene from Camptotheca acuminata, a Camptothecin-Producing Plant

et al. 2015

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Camptothecin (CAM), a complex pentacyclic pyrroloqinoline alkaloid, is the starting material for CAM-type drugs that are well-known antitumor plant drugs. Although many chemical and biological research efforts have been performed to produce CAM, a few attempts have been made to uncover the enzymatic mechanism involved in the biosynthesis of CAM. Enzyme-catalyzed oxidoreduction reactions are ubiquitously presented in living organisms, especially in the biosynthetic pathway of most secondary metabolites such as CAM. Due to a lack of its reduction partner, most catalytic oxidation steps involved in the biosynthesis of CAM have not been established. In the present study, an NADPH-cytochrome P450 reductase (CPR) encoding gene CamCPR was cloned from Camptotheca acuminata, a CAM-producing plant. The full length of CamCPR cDNA contained an open reading frame of 2127-bp nucleotides, corresponding to 708-amino acid residues. CamCPR showed 70 ~ 85% identities to other characterized plant CPRs and it was categorized to the group II of CPRs on the basis of the results of multiple sequence alignment of the N-terminal hydrophobic regions. The intact and truncate CamCPRs with N- or C-terminal His6-tag were heterologously overexpressed in Escherichia coli. The recombinant enzymes showed NADPH-dependent reductase activity toward a chemical substrate ferricyanide and a protein substrate cytochrome c. The N-terminal His6-tagged CamCPR showed 18- ~ 30-fold reduction activity higher than the C-terminal His6-tagged CamCPR, which supported a reported conclusion, i.e., the last C-terminal tryptophan of CPRs plays an important role in the discrimination between NADPH and NADH. Co-expression of CamCPR and a P450 monooxygenase, CYP73A25, a cinnamate 4-hydroxylase from cotton, and the following catalytic formation of p-coumaric acid suggested that CamCPR transforms electrons from NADPH to the heme center of P450 to support its oxidation reaction. Quantitative real-time PCR analysis showed that CamCPR was expressed in the roots, stems, and leaves of C. acuminata seedlings. The relative transcript level of CamCPR in leaves was 2.2-fold higher than that of roots and the stems showed 1.5-fold transcript level higher than the roots. The functional characterization of CamCPR will be helpful to disclose the mysterious mechanisms of the biosynthesis of CAM. The present study established a platform to characterize the P450 enzymes involved in the growth, development, and metabolism of eukaryotic organisms.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Molecular Cloning, Heterologous Expression, and Functional Characterization of an NADPH-Cytochrome P450 Reductase Gene from Camptotheca acuminata, a Camptothecin-Producing Plant

et al. 2015

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“…Class BmCPR is known to transfer electrons to Etp1 fd and various Fds but also supports the reaction of the microsomal CYP21A2 very efficiently without any Fd mediator [134,135]. In contrast, CaCPR1 supports activity with microsomal CYP1A2 only at low rates relative to the cognate mammalian cytochrome P450 reductase [136]. Given the ability of BmCPR to support CYP21A2 reactions with high efficiency, BmCPR appears to be a more promising thermostable redox partner to support other microsomal P450-catalyzed reactions.…”

Section: Thermostable Diflavin Reductasesmentioning

confidence: 99%

“…Neither BmCPR nor CaCPR1 have been crystallized and therefore the factors responsible for the stability of these forms have not yet been elucidated. However, in the case of the plant CaCPR1, it was speculated that conditions of stress during plant development may have imposed selection pressure to improve the supply electrons to P450s, leading to improved stability [136].…”

Section: Thermostable Diflavin Reductasesmentioning

confidence: 99%

Determinants of thermostability in the cytochrome P450 fold

Harris

Thomson

Strohmaier

et al. 2018

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Cytochromes P450 are found throughout the biosphere in a wide range of environments, serving a multitude of physiological functions. The ubiquity of the P450 fold suggests that it has been co-opted by evolution many times, and likely presents a useful compromise between structural stability and conformational flexibility. The diversity of substrates metabolized and reactions catalyzed by P450s makes them attractive starting materials for use as biocatalysts of commercially useful reactions. However, process conditions impose different requirements on enzymes to those in which they have evolved naturally. Most natural environments are relatively mild, and therefore most P450s have not been selected in Nature for the ability to withstand temperatures above ~40°C, yet industrial processes frequently require extended incubations at much higher temperatures. Thus, there has been considerable interest and effort invested in finding or engineering thermostable P450 systems. Numerous P450s have now been identified in thermophilic organisms and analysis of their structures provides information as to mechanisms by which the P450 fold can be stabilized. In addition, protein engineering, particularly by directed or artificial evolution, has revealed mutations that serve to stabilize particular mesophilic enzymes of interest. Here we review the current understanding of thermostability as it applies to the P450 fold, gleaned from the analysis of P450s characterized from thermophilic organisms and the parallel engineering of mesophilic forms for greater thermostability. We then present a perspective on how this information might be used to design stable P450 enzymes for industrial application. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.

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“…[5] Recently, the resurrected ancestors of microsomal xenobiotic-metabolizing P450s were shown to be highly thermostable, while often retaining similar substrate specificity to the extant forms, properties that can be harnessed for biocatalysis [6] (Harris et al unpublished, Thomson et al, unpublished). However, like their extant descendants, they rely on a redox partner, being routinely supported by the human cytochrome P450 reductase (hCPR), which is thermolabile [7] (Harris Strohmaier et al, unpublished). A potential alternative solution is to take advantage of the peroxide shunt, a means of short-circuiting the P450 catalytic cycle.…”

Section: Introductionmentioning

confidence: 99%

An Inexpensive, Efficient Alternative to NADPH to Support Catalysis by Thermostable Cytochrome P450 Enzymes

et al. 2020

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Cytochrome P450 enzymes (P450s) are highly desirable catalysts for the regio‐ and stereo‐selective, late‐stage functionalization of pharmaceuticals and other fine chemicals. Recently, the resurrected ancestors of drug‐metabolizing P450s were shown to be highly thermostable and expressed in high yield, while retaining similar substrate specificity to the extant forms. However, they still rely on NADPH and cytochrome P450 reductase (CPR) to enable catalysis of oxidative transformations. To identify an alternative support system, we screened 10 oxygen surrogates (OSs) for the ability to support P450 ancestors from three different families. Of the 23 ancestors examined, 17 were supported by at least one OS as well as, or better than, by CPR. Using two candidate P450s we showed that OS‐dependent P450 catalysis can be optimized in a few steps, boosting product yield from ∼2.2 % with CPR to 88–100 % with an OS. The principles applied here will facilitate faster evaluation and optimization of OS‐supported P450 catalysis versus redox partner‐dependent P450 catalysis.

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Heterologous expression and functional characterization of the NADPH-cytochrome P450 reductase from Capsicum annuum

Cited by 20 publications

References 27 publications

Molecular Cloning, Heterologous Expression, and Functional Characterization of an NADPH-Cytochrome P450 Reductase Gene from Camptotheca acuminata, a Camptothecin-Producing Plant

Molecular Cloning, Heterologous Expression, and Functional Characterization of an NADPH-Cytochrome P450 Reductase Gene from Camptotheca acuminata, a Camptothecin-Producing Plant

Determinants of thermostability in the cytochrome P450 fold

An Inexpensive, Efficient Alternative to NADPH to Support Catalysis by Thermostable Cytochrome P450 Enzymes

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