A Conservative Amino Acid Substitution Alters the Regiospecificity of CYP94A2, a Fatty Acid Hydroxylase from the Plant Vicia sativa

Kahn, Rachel Alice; Bouquin, Renaud Le; Pinot, Franck; Benveniste, Irène; Durst, Francis

doi:10.1006/abbi.2001.2415

Cited by 38 publications

(35 citation statements)

References 38 publications

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“…In contrast, substitution of Phe for Ile at position 479 of CYP3A4 alters the regiospecific hydroxylation of 7-hexylcoumarin (60). Similarly, substitution of smaller hydrophobic amino acids (Ala, Val, and Leu) for Phe 494 in CYP94A2 also alters the regiospecificity of lauric acid hydroxylation (61). Finally, docking putative substrates into molecular models of four phenyl propanoid metabolism P450 enzymes from Arabidopsis thaliana (CYP84A1, CYP75B1, CYP98A3, and CYP73A5) was used to map substrate contact points to positions equivalent to SCHEME 2.…”

Section: -Epiaristolochene 13-dihydroxylasementioning

confidence: 91%

“…SRS-5 and SRS-6 were of particular interest given their proximity to the active site in several P450 structures (51) and because specific amino acid positions within these regions have previously been correlated with regio-and stereospecific reaction mechanisms (Fig. 4A) (52)(53)(54)(55)(56)(57)(58)(59)(60)(61). A homology model of EAH derived from comparison with the mammalian CYP2C5 structure was constructed (Fig.…”

Section: -Epiaristolochene 13-dihydroxylasementioning

confidence: 99%

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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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Section: -Epiaristolochene 13-dihydroxylasementioning

confidence: 91%

Section: -Epiaristolochene 13-dihydroxylasementioning

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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“…Members from this clan have previously been shown to be involved in the hydroxylation reactions of fatty acyl-derived substrates (Kahn et al, 2001;Wellesen et al, 2001;Werck-Reichhart et al, 2002) and therefore genes within this clan may be regarded as candidates for cuticle biosynthetic enzymes. To further narrow the choice of gene candidates, expression data have to be taken into account.…”

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confidence: 99%

The Cytochrome P450 Enzyme CYP96A15 Is the Midchain Alkane Hydroxylase Responsible for Formation of Secondary Alcohols and Ketones in Stem Cuticular Wax of Arabidopsis

et al. 2007

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Most aerial surfaces of plants are covered by cuticular wax that is synthesized in epidermal cells. The wax mixture on the inflorescence stems of Arabidopsis (Arabidopsis thaliana) is dominated by alkanes, secondary alcohols, and ketones, all thought to be formed sequentially in the decarbonylation pathway of wax biosynthesis. Here, we used a reverse-genetic approach to identify a cytochrome P450 enzyme (CYP96A15) involved in wax biosynthesis and characterized it as a midchain alkane hydroxylase (MAH1). Stem wax of T-DNA insertional mutant alleles was found to be devoid of secondary alcohols and ketones (mah1-1) or to contain much lower levels of these components (mah1-2 and mah1-3) than wild type. All mutant lines also had increased alkane amounts, partially or fully compensating for the loss of other compound classes. In spite of the chemical variation between mutant and wild-type waxes, there were no discernible differences in the epicuticular wax crystals on the stem surfaces. Mutant stem wax phenotypes could be partially rescued by expression of wild-type MAH1 under the control of the native promoter as well as the cauliflower mosaic virus 35S promoter. Cauliflower mosaic virus 35S-driven overexpression of MAH1 led to ectopic accumulation of secondary alcohols and ketones in Arabidopsis leaf wax, where only traces of these compounds are found in the wild type. The newly formed leaf alcohols and ketones had midchain functional groups on or next to the central carbon, thus matching those compounds in wild-type stem wax. Taken together, mutant analyses and ectopic expression of MAH1 in leaves suggest that this enzyme can catalyze the hydroxylation reaction leading from alkanes to secondary alcohols and possibly also a second hydroxylation leading to the corresponding ketones. MAH1 expression was largely restricted to the expanding regions of the inflorescence stems, specifically to the epidermal pavement cells, but not in trichomes and guard cells. MAH1-green fluorescent protein fusion proteins localized to the endoplasmic reticulum, providing evidence that both intermediate and final products of the decarbonylation pathway are generated in this subcellular compartment and must subsequently be delivered to the plasma membrane for export toward the cuticle.

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“…Among the fatty acid hydroxylases in plants, only P450s perform hydroxylation reactions at the w position (Duan and Schuler 2005). The catalytic properties of a variety of P450 protein families, including CYP86A of Arabidopsis ), CYP94A of Vicia sativa and tobacco Kahn et al 2001;Le Bouquin et al 2001), CYP94B and CYP94C of V. sativa and Arabidopsis , and CYP92B1 from a petunia hybrid (PetkovaAndonova et al 2002), have been extensively characterized in terms of both the chain lengths of their substrates and their hydroxylation positions. CYP709C1 can hydroxylate fatty acids at the w-1 and w-2 positions.…”

Section: Discussionmentioning

confidence: 99%

Metabolomics for the characterization of cytochromes P450-dependent fatty acid hydroxylation reactions in Arabidopsis

Kai

Hashidzume

Yoshimura

et al. 2009

Plant Biotechnology

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The cytochromes P450 (P450s) of higher plants play crucial roles in both primary metabolism and a wide variety of secondary metabolic processes, such as in the phenylpropanoid, terpenoid, and alkaloid pathways, which produce, for example, lignin monomers, flavonoids, anthocyanins, and species-specific phytoalexins (Schuler and Werck-Reichhart 2003). Plant P450s are also involved in the detoxification of xenobiotic chemicals such as herbicides (Schuler and Werck-Reichhart 2003;Nelson et al. 2004). Both the biosynthesis and degradation of plant growth regulators cannot be completed without the involvement of specific P450 protein families (Schuler and Werck-Reichhart 2003). In general, serious phenotypic changes including dwarfism, abnormal morphology, and differential hormonal responses have been the major driving forces of studies to clarify these P450 functions. A good background of natural product chemistry is also indispensable for the functional characterization of P450s. However, irrespective of the importance of plant P450 functions in both understanding plant biological processes and their possible applications as biocatalysts, no general key technology has been established to clarify the diverse enzymatic properties of P450s of unknown functions. For example, even in Arabidopsis thaliana the physiological functions of more than 70% of the P450 proteins remain unknown (Nelson et al. 2004).To develop a versatile experimental platform for P450 characterization, we streamlined a metabolic profiling system based on Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS). Full-length cDNA clones of several Arabidopsis P450 genes encoding unknown functions were selected for the preparation of recombinant enzymes. Those P450s were constitutively expressed in parallel in a cultured cell system and whole plants. Reverse genetics approaches with T-DNA knockout mutants and RNA interference were also incorporated into this metabolic profiling scheme. Changes in the metabolic profiles of T87 cells resulting from P450 overexpression, if any, were evaluated with a comprehensive metabolomics approach Nakamura et al. 2007;Ohta et al. 2007).Here, we report examples of our metabolic profiling study of the CYP78A and CYP86C genes in Arabidopsis. There are six genes for CYP78A subfamily proteins in Arabidopsis, the catalytic properties of which are unknown. It has been reported that the CYP78A proteins are involved in plant developmental processes. Thus, CYP78A11 has been shown to be the PLASTOCHRON1 in rice, which controls the timing of lateral organ formation from the apical meristem (Miyoshi et al. Abstract Genome sequence analysis has revealed the presence of almost infinite numbers of cytochrome P450 genes in a variety of organisms. To establish a robust experimental platform from which to explore the catalytic potential of those putative P450 proteins, we have developed a comprehensive metabolic profiling system based on Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR/MS) analysis, to...

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A Conservative Amino Acid Substitution Alters the Regiospecificity of CYP94A2, a Fatty Acid Hydroxylase from the Plant Vicia sativa

Cited by 38 publications

References 38 publications

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

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

The Cytochrome P450 Enzyme CYP96A15 Is the Midchain Alkane Hydroxylase Responsible for Formation of Secondary Alcohols and Ketones in Stem Cuticular Wax of Arabidopsis

Metabolomics for the characterization of cytochromes P450-dependent fatty acid hydroxylation reactions in Arabidopsis

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