Cloning, Yeast Expression, and Characterization of the Coupling of Two Distantly Related Arabidopsis thalianaNADPH-Cytochrome P450 Reductases with P450 CYP73A5

Urban, Philippe; Mignotte, Claudia; Kazmaier, Michaël; Delorme, Frédéric; Pompon, Denis

doi:10.1074/jbc.272.31.19176

Cited by 311 publications

(267 citation statements)

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“…After BglII and EcoRI digestion, the fragments were directionally cloned into the expression cassette of pYeDP60 (Pompon et al 1996) digested with BamHI and EcoRI. Saccharomyces cerevisiae strain WAT11 is a derivative from strain W303-1B, in which yeast CPR gene (coding for a NADPH-cytochrome P450 reductase) was replaced by the Arabidopsis ATR1 (Urban et al 1997). WAT11 was transformed using the lithium acetate/single stranded carrier DNA/polyethylene glycol method (Gietz and Woods 2002).…”

Section: Methodsmentioning

confidence: 99%

Isolation and mapping of a C3′H gene (CYP98A49) from globe artichoke, and its expression upon UV-C stress

et al. 2009

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Globe artichoke represents a natural source of phenolic compounds with dicaffeoylquinic acids along with their biosynthetic precursor chlorogenic acid (5-caffeoylquinic acid) as the predominant molecules. We report the isolation and characterization of a full-length cDNA and promoter of a globe artichoke p-coumaroyl ester 3 0 -hydroxylase (CYP98A49), which is involved in both chlorogenic acid and lignin biosynthesis. Phylogenetic analyses demonstrated that this gene belongs to the CYP98 family. CYP98A49 was also heterologously expressed in yeast, in order to perform an enzymatic assay with pcoumaroylshikimate and p-coumaroylquinate as substrates. Real Time quantitative PCR analysis revealed that CYP98A49 expression is induced upon exposure to UV-C radiation. A single nucleotide polymorphism in the CYP98A49 gene sequence of two globe artichoke varieties used for genetic mapping allowed the localization of this gene to linkage group 10 within the previously developed maps.

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Section: Methodsmentioning

confidence: 99%

Isolation and mapping of a C3′H gene (CYP98A49) from globe artichoke, and its expression upon UV-C stress

et al. 2009

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“…-242, -332, -412 and -505 are only found in ATR2. In addition, presence at the ATR2 N-terminus of a poly (Ser/Thr) stretch was very unusual among CPRs and might play a role for a possible and unusual chloroplastic subcellular location of this reductase in plants [15]. Formation of disulfide bounds in chloroplasts was postulated to be a regulation mechanism involved in physiological control of enzyme activities [66,67].…”

Section: Discussionmentioning

confidence: 99%

“…In Arabidopsis thaliana, two distantly related Enzymes. NADPH-cytochrome P-450 reductase (EC 1.6.2.4); cytochrome P-450 (EC 1.14.14.1).reductases (ATR1 and ATR2, 64% amino acid sequence identity) were cloned and may localize in different subcellular fractions [15].All CPRs belong to the same gene superfamily with amino acid sequence similarities ranging from 30% up to 90% between bacteria, yeast, fungi, plants, fish, insects and mammals enzymes [14Ϫ33]. Recently, the X-ray structure of rat CPR was obtained and suggests a four-domain folding [34].…”

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confidence: 99%

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Differential redox and electron‐transfer properties of purified yeast, plant and human NADPH‐cytochrome P‐450 reductases highly modulate cytochrome P‐450 activities

Louérat-Oriou

Perret

Pompon

1998

European Journal of Biochemistry

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Saccharomyces, human and two Arabidopsis (ATR1 and ATR2) NADPH-P-450 reductases were expressed in yeast, purified to homogeneity and used to raise antibodies. Among the P-450-reductases, ATR2 contrasted by its very low FMN affinity and required a thiol-reducing agent for efficient cofactor binding to the FMN-depleted enzyme. Analysis of reductase kinetic properties using artificial acceptors and different salt conditions suggested marked differences between reductases in their FAD and FMN environments and confirmed the unusual properties of the ATR2 FMN-binding domain. Courses of flavin reductions by NADPH were analysed by rapid kinetic studies. The human enzyme was characterized by a FAD reduction rate sixfold to tenfold slower than values for the three other reductases. Following the fast phase of reduction, expected accumulation of flavin semiquinone was observed for the human and ATR1 but not for ATR2 and the yeast reductases. Consistently, redox potential for the FMN semiquinone/ reduced couple in the yeast enzyme was found to be more positive than the value for the FMN oxidized/ semiquinone couple. This situation was reminiscent of similar inversion observed in bacterial P-450 BM3 reductase. Affinities of reductases for rabbit P-450 2B4 and supported monooxygenase activities in reconstituted systems highly depended on the reductase source. The human enzyme exhibited the highest affinity but supported the lowest k cat whereas the yeast reductase gave the best k cat but with the lowest affinity. ATR1 exhibited both high affinity and efficiency. No simple relation was found between reductase activies with artificial and natural (P-450) acceptors. Thus marked differences in kinetic and redox parameters between reductases dramatically affect their respective abilities to to support P-450 functions.Keywords : NADPH-cytochrome P-450 reductase; redox potential ; kinetic property; P-450 interaction.NADPH-P-450 reductase (CPR; NADPH : ferrihemoprotein reductase) is a membrane-anchored protein that catalyzes electron transfers from NADPH to cytochrome P-450 (P-450) [1Ϫ4], heme oxygenase, cytochrome b 5 and squalene epoxidase [5,6]. The various members of the P-450 superfamily [7] are involved in CPR-supported-oxidative metabolism of a wide range of xenobiotics and endogenous compounds [8]. Contrasting with the large variety of P-450s, a single CPR is generally present in each organism [9,10]. Unlike animals and yeast, higher plants express multiple forms of CPRs as inferred from western blot analysis of purified materials [11,12]. Durst et al.[13] have isolated two distinct partial length cDNA from Helianthus tuberosus, corresponding to distinct mRNA species. The two CPRs isolated from parsley were 80% identical in amino acid sequence and a distinct metabolic role was clearly assigned to each other [14]. In Arabidopsis thaliana, two distantly related Enzymes. NADPH-cytochrome P-450 reductase (EC 1.6.2.4); cytochrome P-450 (EC 1.14.14.1).reductases (ATR1 and ATR2, 64% amino acid sequence identity) were cloned and may ...

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“…Yeast Expression and Microsome Preparations-Expression vectors harboring wild-type or mutant EAH were introduced into the WAT11 yeast strain, a strain previously engineered with an Arabidopsis NADPH-cytochrome P450 reductase gene (40) and kindly provided by Dr. P. Urban (Centre de Génétique Moléculaire, CNRS, Gif-sur-Yvette, France). Transformants were grown from single colonies; expression of the EAH cDNAs was induced by galactose addition to the growth medium; and microsomes were prepared as described previously (41).…”

Section: Methodsmentioning

confidence: 99%

Kinetic and Molecular Analysis of 5-Epiaristolochene 1,3-Dihydroxylase, a Cytochrome P450 Enzyme Catalyzing Successive Hydroxylations of Sesquiterpenes

Takahashi

Zhao

O’Maille

et al. 2005

Journal of Biological Chemistry

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The final step of capsidiol biosynthesis is catalyzed by 5-epiaristolochene dihydroxylase (EAH), a cytochrome P450 enzyme that catalyzes the regio-and stereospecific insertion of two hydroxyl moieties into the bicyclic sesquiterpene 5-epiaristolochene (EA). Detailed kinetic studies using EA and the two possible monohydroxylated intermediates demonstrated the release of 1␤-hydroxy-EA ((OH)EA) at high EA concentrations and a 10-fold catalytic preference for 1␤(OH)EA versus 3␣(OH)EA, indicative of a preferred reaction order of hydroxylation at C-1, followed by that at C-3. Sequence alignments and homology modeling identified activesite residues tested for their contribution to substrate specificity and overall enzymatic activity. Mutants EAH-S368C and EAH-S368V exhibited wild-type catalytic efficiencies for 1␤(OH)EA biosynthesis, but were devoid of the successive hydroxylation activity for capsidiol biosynthesis. In contrast to EAH-S368C, EAH-S368V catalyzed the relative equal biosynthesis of 1␤(OH)EA, 2␤(OH)EA, and 3␤(OH)EA from EA with wild-type efficiency. Moreover, EAH-S368V converted ϳ1.5% of these monohydroxylated products to their respective ketone forms. Alanine and threonine mutations at position 368 were significantly compromised in their conversion rates of EA to capsidiol and correlated with 3.6-and 5.7-fold increases in their K m values for the 1␤(OH)EA intermediate, respectively. A role for Ile 486 in the successive hydroxylations of EA was also suggested by the EAH-I468A mutant, which produced significant amounts 1␤(OH)EA, but negligible amounts of capsidiol from EA. The altered product profile of the EAH-I486A mutant correlated with a 3.6-fold higher K m for EA and a 4.4-fold slower turnover rate (k cat ) for 1␤(OH)EA. These kinetic and mutational studies were correlated with substrate docking predictions to suggest how Ser 368 and Ile 486 might contribute to active-site topology, substrate binding, and substrate presentation to the oxo-Fe-heme reaction center.The mevalonate and methylerythritol phosphate pathways are responsible for the biosynthesis of isoprenoids, a diverse group of organic natural products found in animals, plants, fungi, insects, and bacteria. Isoprenoids are further divided into classes of primary and secondary metabolites. Isoprenoids that are primary metabolites include sterols, carotenoids, hormones, and long chain hydrocarbons used to tether particular enzymes to membrane systems, compounds essential for viability. Isoprenoids classified as secondary metabolites include monoterpenes, sesquiterpenes, diterpenes, and triterpenes, and many of these mediate interactions between organisms and their environments (1-5).Capsidiol is a bicyclic dihydroxylated sesquiterpene produced by several solanaceous plants in response to pathogen or elicitor challenge (6 -10) and is considered an important plant defense response because it can prevent the germination and growth of several fungal species (11). Capsidiol biosynthesis is regulated by the expression of two key enzymes (see Scheme 1...

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Cloning, Yeast Expression, and Characterization of the Coupling of Two Distantly Related Arabidopsis thalianaNADPH-Cytochrome P450 Reductases with P450 CYP73A5

Cited by 311 publications

References 61 publications

Isolation and mapping of a C3′H gene (CYP98A49) from globe artichoke, and its expression upon UV-C stress

Isolation and mapping of a C3′H gene (CYP98A49) from globe artichoke, and its expression upon UV-C stress

Differential redox and electron‐transfer properties of purified yeast, plant and human NADPH‐cytochrome P‐450 reductases highly modulate cytochrome P‐450 activities

Kinetic and Molecular Analysis of 5-Epiaristolochene 1,3-Dihydroxylase, a Cytochrome P450 Enzyme Catalyzing Successive Hydroxylations of Sesquiterpenes

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