A conformationally restricted uniconazole analogue as a specific inhibitor of rice ent-kaurene oxidase, CYP701A6

The plant growth retardant uniconazole (UNI), which has been used as an effective inhibitor of ent-kaurene oxidase (CYP701A) involved in gibberellin biosynthesis, also strongly inhibits ABA 8'-hydroxylase (CYP707A), a key enzyme in abscisic acid catabolism. Azole P450 inhibitors bind to the P450 active site by both coordinating to the heme-iron atom via an sp(2) nitrogen and interacting with surrounding protein residues through a lipophilic region. We hypothesized that poor selectivity of UNI may result from its small molecular size and flexible conformation that allows it to fit into active sites differing in size and shape. To find a selective inhibitor of CYP701A based on this hypothesis, we examined inhibitory activities of three types of UNI analogues, which were conformationally constrained, enlarged in width, and enlarged in length, against recombinant rice CYP701A6 and Arabidopsis CYP707A3. Conformationally restricted analogues, UFAP2 and UFAP2N, inhibited CYP701A6 as strongly as UNI, whereas it inhibited CYP707A3 less than UNI.     

Abscinazole-E1, a novel chemical tool for exploring the role of ABA 8'-hydroxylase CYP707A

We developed abscinazole-E1 (Abz-E1), a specific inhibitor of abscisic acid (ABA) 8′-hydroxylase (CYP707A). This inhibitor was designed and synthesized as an enlarged analogue of uniconazole (UNI), a well-known plant growth retardant, which inhibits a gibberellin biosynthetic enzyme (ent-kaurene oxidase, CYP701A) as well as CYP707A. Our results showed that Abz-E1 functions as a potent inhibitor of CYP707A and a poor inhibitor of CYP701A both in vitro and in vivo. Abz-E1 application to plants resulted in improved desiccation tolerance and an increase in endogenous ABA.     

ABA 9'-hydroxylation is catalyzed by CYP707A in Arabidopsis

Abscisic acid (ABA) catabolism is important for regulating endogenous ABA levels. To date, most effort has focused on catabolism of ABA to phaseic acid (PA), which is generated spontaneously after 8′-hydroxylation of ABA by cytochrome P450s in the CYP707A subfamily. Neophaseic acid (neoPA) is another well-documented ABA catabolite that is produced via ABA 9′-hydroxylation, but the 9′-hydroxylase has not yet been defined. Here, we show that endogenous neoPA levels are reduced in loss-of-function mutants defective in CYP707A genes. In addition, in planta levels of both neoPA and PA are reduced after treatment of plants with uniconazole-P, a P450 inhibitor. These lines of evidence suggest that CYP707A genes also encode the 9′-hydroxylase required for neoPA synthesis. To test this, in vitro enzyme assays using microsomal fractions from CYP707A-expressing yeast strains were conducted and these showed that all four Arabidopsis CYP707As are 9′-hydroxylases, although this activity is minor. Collectively, our results demonstrate that ABA 9′-hydroxylation is catalyzed by CYP707As as a side reaction.     

The Arabidopsis cytochrome P450 CYP707A encodes ABA 8'-hydroxylases: key enzymes in ABA catabolism

The hormonal action of abscisic acid (ABA) in plants is controlled by the precise balance between its biosynthesis and catabolism. In plants, ABA 8'-hydroxylation is thought to play a predominant role in ABA catabolism. ABA 8'-hydroxylase was shown to be a cytochrome P450 (P450); however, its corresponding gene had not been identified. Through phylogenetic and DNA microarray analyses during seed imbibition, the candidate genes for this enzyme were narrowed down from 272 Arabidopsis P450 genes. These candidate genes were functionally expressed in yeast to reveal that members of the CYP707A family, CYP707A1-CYP707A4, encode ABA 8'-hydroxylases. Expression analyses revealed that CYP707A2 is responsible for the rapid decrease in ABA level during seed imbibition. During drought stress conditions, all CYP707A genes were upregulated, and upon rehydration a significant increase in mRNA level was observed. Consistent with the expression analyses, cyp707a2 mutants exhibited hyperdormancy in seeds and accumulated six-fold greater ABA content than wild type. These results demonstrate that CYP707A family genes play a major regulatory role in controlling the level of ABA in plants.     

Abscinazole-E2B, a practical and selective inhibitor of ABA 8'-hydroxylase CYP707A

We developed abscinazole-E2B (Abz-E2B), a practical and specific inhibitor of abscisic acid (ABA) 8'-hydroxylase (CYP707A), by structural modification of abscinazole-E1 (Abz-E1), another compound we developed. A butoxy group was introduced to Abz-E2B instead of the tosylate group of Abz-E1, in expectation of better water solubility, because the calculated logP value of Abz-E2B is 3.47, which is smaller than that of Abz-E1 (4.02). The water solubility of Abz-E2B was greater than 90% at a concentration of 100 μM, at which the solubility of Abz-E1 was 20%. The enzyme specificity was improved significantly. In in vitro assays constructed using recombinant enzymes, (±)-Abz-E2B was a considerably weaker inhibitor than (±)-Abz-E1 for CYP701A, a GA biosynthetic enzyme, which is a target of S-uniconazole (S-UNI), a lead compound of Abz-E1. (±)-Abz-E2B application to plants resulted in improved desiccation tolerance and an increase in endogenous ABA, with little retardation of growth. We also prepared optically pure Abz-E2B and determined its absolute configuration. The R-enantiomer of Abz-E2B was the more potent inhibitor of CYP707A, unlike UNI, whereas both enantiomers were markedly less effective than S-UNI in inhibiting CYP701A. Because S-Abz-E2B arrested the growth of rice seedlings at 100 μM, probably because of off-target effects, R-Abz-E2B should be used as a chemical tool for research focusing on CYP707A when 100 μM or higher concentration is required, although (±)-Abz-E2B may be useful as an alternative option at a lower concentration.     

CYP707A3, a major ABA 8'-hydroxylase involved in dehydration and rehydration response in Arabidopsis thaliana

Abscisic acid (ABA) catabolism is one of the determinants of endogenous ABA levels affecting numerous aspects of plant growth and abiotic stress responses. The major ABA catabolic pathway is triggered by ABA 8'-hydroxylation catalysed by the cytochrome P450 CYP707A family. Among four members of Arabidopsis CYP707As, the expression of CYP707A3 was most highly induced in response to both dehydration and subsequent rehydration. A T-DNA insertional cyp707a3-1 mutant contained higher ABA levels in turgid plants, which showed a reduced transpiration rate and hypersensitivity to exogenous ABA during early seedling growth. On dehydration, the cyp707a3-1 mutant accumulated a higher amount of stress-induced ABA than the wild type, an event that occurred relatively later and was coincident with slow drought induction of CYP707A3. The cyp707a3 mutant plants exhibited both exaggerated ABA-inducible gene expression and enhanced drought tolerance. Conversely, constitutive expression of CYP707A3 relieved growth retardation by ABA, increased transpiration, and a reduction of endogenous ABA in both turgid and dehydrated plants. Taken together, our results indicate that CYP707A3 plays an important role in determining threshold levels of ABA during dehydration and after rehydration.     

ABA分解代谢及其代谢关键酶——8'-羟化酶

脱落酸(ABA)在植物的生长发育和环境胁迫响应等过程中具有重要作用。ABA合成与分解代谢的动态平衡共同调控植物内源ABA水平。ABA8′位甲基羟基化途径是高等植物内源ABA代谢的主要途径;8′-羟化酶是该代谢途径的关键酶,属于P450酶系。生物化学和基因组学研究表明,拟南芥CYP707A家族基因编码8′-羟化酶,该基因家族广泛存在于高等植物中,调控植物内源ABA代谢,介导ABA相关的生理生化过程。本文综述了ABA分解代谢的基本途径,详细概述了ABA8′位甲基羟基化途径及该代谢途径的关键酶8′-羟化酶。同时介绍了8′-羟化酶编码基因-CYP707A家族基因的生物学特征和功能。     

Asymmetrical ligand binding by abscisic acid 8'-hydroxylase

Abscisic acid (ABA), a plant stress hormone, has a chiral center (C1') in its molecule, yielding the enantiomers (1'S)-(+)-ABA and (1'R)-(-)-ABA during chemical synthesis. ABA 8'-hydroxylase (CYP707A), which is the major and key P450 enzyme in ABA catabolism in plants, catalyzes naturally occurring (1'S)-(+)-enantiomer, whereas it does not recognize naturally not occurring (1'R)-(-)-enantiomer as either a substrate or an inhibitor. Here we report a structural ABA analogue (AHI1), whose both enantiomers bind to recombinant Arabidopsis CYP707A3, in spite of stereo-structural similarity to ABA. The difference of AHI1 from ABA is the absence of the side-chain methyl group (C6) and lack of the alpha,beta-unsaturated carbonyl (C2'C3'-C4'O) in the six-membered ring. To explore which moiety is responsible for asymmetrical binding by CYP707A3, we synthesized and tested ABA analogues that lacked each moiety. Competitive inhibition was observed for the (1'R) enantiomers of these analogues in the potency order of (1'R,2'R)-(-)-2',3'-dihydro-4'-deoxo-ABA (K(I)=0.45 microM)>(1'R)-(-)-4'-oxo-ABA (K(I)=27 microM)>(1'R)-(-)-6-nor-ABA and (1'R,2'R)-(-)-2',3'-dihydro-ABA (no inhibition). In contrast to the (1'R)-enantiomers, the inhibition potency of the (1'S)-analogues declined with the saturation of the C2',C3'-double bond or with the elimination of the C4'-oxo moiety. These findings suggest that the C4'-oxo moiety coupled with the C2',C3'-double bond is the significant key functional group by which ABA 8'-hydroxylase distinguishes (1'S)-(+)-ABA from (1'R)-(-)-ABA.     

Structural Basis of Drug Binding to CYP46A1, an Enzyme That Controls Cholesterol Turnover in the Brain

Cytochrome P450 46A1 (CYP46A1) initiates the major pathway of cholesterol elimination from the brain and thereby controls cholesterol turnover in this organ. We determined x-ray crystal structures of CYP46A1 in complex with four structurally distinct pharmaceuticals; antidepressant tranylcypromine (2.15 Å), anticonvulsant thioperamide (1.65 Å), antifungal voriconazole (2.35 Å), and antifungal clotrimazole (2.50 Å). All four drugs are nitrogen-containing compounds that have nanomolar affinity for CYP46A1 in vitro yet differ in size, shape, hydrophobicity, and type of the nitrogen ligand. Structures of the co-complexes demonstrate that each drug binds in a single orientation to the active site with tranylcypromine, thioperamide, and voriconazole coordinating the heme iron via their nitrogen atoms and clotrimazole being at a 4 Å distance from the heme iron. We show here that clotrimazole is also a substrate for CYP46A1. High affinity for CYP46A1 is determined by a set of specific interactions, some of which were further investigated by solution studies using structural analogs of the drugs and the T306A CYP46A1 mutant. Collectively, our results reveal how diverse inhibitors can be accommodated in the CYP46A1 active site and provide an explanation for the observed differences in the drug-induced spectral response. Co-complexes with tranylcypromine, thioperamide, and voriconazole represent the first structural characterization of the drug binding to a P450 enzyme.     

Differences between the structural requirements for ABA 8'-hydroxylase inhibition and for ABA activity

A major catabolic enzyme of the plant hormone abscisic acid (ABA) is the cytochrome P450 monooxygenase ABA 8'-hydroxylase. For designing a specific inhibitor of this enzyme, the substrate specificity and inhibition of CYP707A3, an ABA 8'-hydroxylase from Arabidopsis thaliana, was investigated using 45 structural analogues of ABA and compared to the structural requirements for ABA activity. Substrate recognition by the enzyme strictly required the 6'-methyl groups (C-8' and C-9'), which were unnecessary for ABA activity, whereas elimination of the 3-methyl (C-6) and 1'-hydroxyl groups, which significantly affected ABA activity, had little effect on the ability of analogues to competitively inhibit the enzyme. Fluorination at C-8' and C-9' resulted in resistance to 8'-hydroxylation and competitive inhibition of the enzyme. In particular, 8',8'-difluoro-ABA and 9',9'-difluoro-ABA yielded no enzyme reaction products and strongly inhibited the enzyme (K(I) = 0.16 and 0.25 microM, respectively).     

Effect of the minor ABA metabolite 7'-hydroxy-ABA on Arabidopsis ABA 8'-hydroxylase CYP707A3

To examine the effect of the minor abscisic acid (ABA) metabolite 7'-hydroxy-ABA on Arabidopsis ABA 8'-hydroxylase (CYP707A3), we developed a novel and facile, four-step synthesis of 7'-hydroxy-ABA from alpha-ionone. Structural analogues of 7'-hydroxy-ABA, 1'-deoxy-7'-hydroxy-ABA, and 7'-oxo-ABA were also synthesized to evaluate the role of the 7'-hydroxyl group on binding to the enzyme. The result of enzyme inhibition assay suggests that the local polarity at C-7', neither steric bulkiness nor overall molecular hydrophilicity, would be the major reason why (+)-7'-hydroxy-ABA is not a potent inhibitor of CYP707A3.     

Differential inhibition of Candida albicans CYP51 with azole antifungal stereoisomers

Azole antifungal compounds are important in agriculture and in the treatment of mycotic infection The target enzyme, sterol 14α-demethylase (CYP51), is inhibited through binding of triazole N-4 to the haem of this P450, as a sixth ligand together with the N-1 substituent groups interacting in some way with the apoprotein. Here we use Saccharomyces cerevisiae expression systems for the target enzyme of Candida albicans to investigate binding of enantiomers of the azole antifungal compounds SCH39304 and tetraconazole. A molecular model produced previously provided qualitative explanations for these differences. Interaction of the azole antifungal aromatic group with Phe-233 or -235 may cause the higher activity for (R)-tetraconazole while inactivity of the (SS)-enantiomer of SCH39304 was predicted to result from incompatibility of the hydrophilic sulfonyl moiety when located into the hydrophobic pocket of the active site.     

Characterization of genes encoding ABA 8'-hydroxylase in ethylene-induced stem growth of deepwater rice (Oryza sativa L.)

Ethylene and submergence enhance stem elongation of deepwater rice, at least in part, by reducing in the internode the endogenous abscisic acid (ABA) content and increasing the level of gibberellin A1 (GA1). We cloned and characterized the CYP707A5 and CYP707A6 genes, which encode putative ABA 8'-hydroxylase, the enzyme that catalyzes the oxidation of ABA. Expression of CYP707A5 was upregulated significantly by ethylene treatment, whereas that of CYP707A6 was not altered. Recombinant proteins from both genes expressed in yeast cells showed activity of ABA 8'-hydroxylase. This finding indicates that CYP707A5 may play a role in ABA catabolism during submergence- or ethylene-induced stem elongation in deepwater rice. Taken together, these results provide links between the molecular mechanisms and physiological phenomena of submergence- and ethylene-induced stem elongation in deepwater rice.     

Expression of ABA 8'-hydroxylases in relation to leaf water relations and seed development in bean

In plants, the level of abscisic acid (ABA) is determined by synthesis and catabolism. Hydroxylation of ABA at the 8' position is the key step in ABA catabolism. This reaction is catalyzed by ABA 8'-hydroxylase, a cytochrome P450 (CYP). The cDNAs of PvCYP707A1 and PvCYP707A2 were isolated from bean (Phaseolus vulgaris L.) axes treated with (+)-ABA and that of PvCYP707A3 from dehydrated bean leaves. The recombinant PvCYP707A proteins expressed in yeast were biochemically characterized. Yeast strains over-expressing any of the three PvCYP707As were able to convert ABA to phaseic acid (PA). The microsomal fractions from these yeast strains also exhibited ABA 8'-hydroxylase activity. Expression of PvCYP707A3 in primary leaves was strongly increased by water stress, whereas PvCYP707A1 and PvCYP707A2 mRNA levels were rapidly increased by rehydration of water-stressed leaves. Northern blot analysis of PvCYP707As in bean showed a high level of expression in the mature fruits, senescent leaves, roots, seed coats and axes. All three PvCYP707As were expressed at varying intensities throughout seed development. Imbibed seeds also had high PvCYP707A mRNA levels. Thus, expression of PvCYP707As is both environmentally and developmentally regulated. Transgenic Nicotiana sylvestris plants over-expressing PvCYP707As displayed a wilty phenotype, and had reduced ABA levels and increased PA levels. These results demonstrate that expression of PvCYP707As is the major mechanism by which ABA catabolism is regulated in bean.     

Differences in flexibility of active sites of cytochromes P450 probed by resonance Raman and UV-Vis absorption spectroscopy.

Spectroscopic methods reveal differences in flexibility and stability of P450 forms. Among microsomal P450s, the most flexible active site has been found in the CYP3A4 enzyme as it is compressible and the heme vinyl side chains may adopt two different conformations. On the other hand, active site of this enzyme denatures quite easily upon hydrostatic pressure. The most rigid active site able to withstand the effect of high pressure has CYP1A2. The bacterial CYP102 (BM3) flavocytochrome has also a rather stable, but flexible active site. The differences between CYP3A4 and CYP1A2 active sites apparently reflect their ability to bind various substrates: whereas the CYP3A4 binds a vast variety of structures, the CYP1A2 preferentially binds planar, aromatic structures and its substrate specificity is relatively narrow.     

Seed dormancy and ABA metabolism in Arabidopsis and barley: the role of ABA 8'-hydroxylase

We have investigated the relationship between seed dormancy and abscisic acid (ABA) metabolism in the monocot barley and the dicot Arabidopsis. Whether dormant (D) or non-dormant (ND), dry seed of Arabidopsis and embryos of dry barley grains all had similarly high levels of ABA. ABA levels decreased rapidly upon imbibition, although they fell further in ND than in D. Gene expression profiles were determined in Arabidopsis for key ABA biosynthetic [the 9-cis epoxycarotenoid dioxygenasegene family] and ABA catabolic [the ABA 8'-hydroxylase gene family (CYP707A)] genes. Of these, only the AtCYP707A2 gene was differentially expressed between D and ND seeds, being expressed to a much higher level in ND seeds. Similarly, a barley CYP707 homologue, (HvABA8'OH-1) was expressed to a much higher level in embryos from ND grains than from D grains. Consistent with this, in situ hybridization studies showed HvABA8'OH-1 mRNA expression was stronger in embryos from ND grains. Surprisingly, the signal was confined in the coleorhiza, suggesting that this tissue plays a key role in dormancy release. Constitutive expression of a CYP707A gene in transgenic Arabidopsis resulted in decreased ABA content in mature dry seeds and a much shorter after-ripening period to overcome dormancy. Conversely, mutating the CYP707A2 gene resulted in seeds that required longer after-ripening to break dormancy. Our results point to a pivotal role for the ABA 8'-hydroxylase gene in controlling dormancy and that the action of this enzyme may be confined to a particular organ as in the coleorhiza of cereals.