Eur. J. Biochem. 267, 3661±3671 (2000) q FEBS 2000
A novel NADPH:diamide oxidoreductase activity in Arabidopsis thaliana P1 z-crystallin Jun'ichi Mano1, Elena Babiychuk2, Enric Belles-Boix2, Jun Hiratake3, Akira Kimura1, Dirk InzeÂ2, Sergei Kushnir2 and Kozi Asada4 1
Research Institute for Food Science, Kyoto University, Uji, Japan; 2Department of Genetics, Flanders Interuniversity Institute for Biotechnology, University of Ghent, Belgium; 3Institute for Chemical Research, Kyoto University, Uji, Japan; 4Department of Biotechnology, Faculty of Engineering, Fukuyama University, Japan
The z-crystallin (ZCr) gene P1 of Arabidopsis thaliana, known to confer tolerance toward the oxidizing drug 1,1 0 -azobis(N,N-dimethylformamide) (diamide) to yeast [Babiychuk, E., Kushnir, S., Belles-Boix, E., Van Montagu, M. & InzeÂ, D. (1995) J. Biol. Chem. 270, 26224], was expressed in Escherichia coli to characterize biochemical properties of the P1-z-crystallin (P1-ZCr). Recombinant P1-ZCr, a noncovalent dimer, showed NADPH:quinone oxidoreductase activity with specificity to quinones similar to that of guinea-pig ZCr. P1-ZCr also catalyzed the divalent reduction of diamide to 1,2-bis(N,N-dimethylcarbamoyl)hydrazine, with a k cat comparable with that for quinones. Two other azodicarbonyl compounds also served as substrates of P1-ZCr. Guinea-pig ZCr, however, did not catalyze the azodicarbonyl reduction. Hence, plant ZCr is distinct from mammalian ZCr, and can be referred to as NADPH:azodicarbonyl/quinone reductase. The quinone-reducing reaction was accompanied by radical chain reactions to produce superoxide radicals, while the azodicarbonylreducing reaction was not. Specificity to NADPH, as judged by k cat /Km, was . 1000-fold higher than that to NADH both for quinones and diamide. N-Ethylmaleimide and p-chloromercuribenzoic acid inhibited both quinone-reducing and diamide-reducing activities. Both NADPH and NADP1 suppressed the inhibition, but NADH did not, suggesting that sulfhydryl groups reside in the binding site for the phosphate group on the adenosine moiety of NADPH. The diamide-reducing activity of P1-ZCr accounts for the tolerance of P1-overexpressing yeast to diamide. Other possible physiological functions of P1-ZCr in plants are discussed. Keywords: Arabidopsis thaliana; diamide; P1 protein; quinone reductase; z-crystallin.
Production of O2 2 and H2O2 via partial reduction of dioxygen inevitably accompanies respiration, photosynthesis and various redox reactions in cells. These species and their derivatives, the more reactive hydroxyl radicals, thiyl radicals and lipid radicals, impair membranes, proteins, DNA and carbohydrates [1]. As the primary defense against this oxidative stress in normal metabolism, cells are equipped with antioxidants, such as ascorbate, glutathione, b-carotene, a-tocopherol, and also specific scavenging enzymes, such as superoxide dismutase (SOD) and catalase to disproportionate O2 and H2O2, 2 respectively, and glutathione peroxidase in mammals and ascorbate peroxidase in plants to reduce H2O2 to H2O. Under various environmental stresses, such as drought, low temperature, UV irradiation and xenobiotics, oxidative stress is enhanced in plants [2±4], mainly due to an overflow of excess Correspondence to J. Mano, Research Institute for Food Science, Kyoto University, Uji, 611-0011 Japan. Fax: 1 81 774 33 3004, Tel.: 1 81 774 38 3731, E-mail:
[email protected] Abbreviations: diamide, 1,1 0 -azobis(N, N-dimethylformamide); dipiperidine, 1,1 0 -(azodicarbonyl)dipiperidine; DPQ, decyl-plastoquinone; HRMS, high resolution mass spectrometry; hydrazodicarbonamide, 1,2-bis(N,N-dimethylcarbamoyl)hydrazine; P1-ZCr, the Arabidopsis thaliana P1-z-crystallin; PAQ, 9,10-phenanthrenequionone; QOR, quinone oxidoreductase; SOD, superoxide dismutase; ZCr, z-crystallin. Note: a web page is available at: http://www.food.food.kyoto-u.ac.jp/bunya12/KIMURA/index_e.html (Received 29 February 2000, accepted 12 April 2000)
light energy that is not used for CO2 assimilation [5]. A number of defense genes, some of which have potential antioxidant functions, are induced by such stresses [3,6], but not all have been identified. A useful approach for finding new antioxidant genes is heterologous expression screening, in which plant genes that improve the tolerance of bacteria or yeast towards oxidative stress are cloned [7,8]. By conferring tolerance toward the oxidizing drug 1,1 0 -azobis(N,N-dimethylformamide) (diamide) to Saccharomyces cerevisiae, we cloned several new genes from a cDNA library of Arabidopsis thaliana. One of these, the glutathione S-transferase gene was induced by oxidative stress in A. thaliana, supporting the hypothesis that this gene functions under stress [7]. Interestingly, when we used the yap1D mutant of S. cerevisiae, which is sensitive to oxidative stress because of a deficiency in the oxidative stressresponsive activation factor yAP1, a completely different set of genes was cloned from the same A. thaliana cDNA library [9]. This suggested that there are several mechanisms which protect cells from diamide toxicity. Three of the four newly isolated genes, named P1, P2 and P4, were identified as z-crystallin (ZCr) homologs. The P1 gene was induced in A. thaliana by various oxidative-stress treatments, suggesting that the product of P1 gene, P1-z-crystallin (P1-ZCr), may be involved in an unknown antioxidative mechanism in plants. The deduced amino-acid sequence of P1-ZCr is 25% identical and has 50% residue similarity to that of ZCr in guinea pigs [9]. ZCr is a major lens protein (< 10% of total protein) found in hystricomorph rodents and camelids [10].
3662 J. Mano et al. (Eur. J. Biochem. 267)
Genetic deficiency of ZCr causes congenital cataract in guinea pigs, suggesting a protective function of ZCr against deterioration of the lens cells [11,12]. ZCr in guinea pigs and camels catalyzes the NADPH-dependent reduction of artificial quinones [13±15], and it has been proposed that ZCr activates redox cycling of NADPH and glutathione on xenobiotic stress [16]. However, the biochemical reactions that contribute to lens protection have yet to be identified. ZCr homologs are widely distributed from bacteria to mammals [17] and higher plants [18], and commonly possess NADPH-binding sites, with the exception of that from the bovine lens [19]. Some have been characterized as dehydrogenases or reductases. ZCr homologs share a conserved three-dimensional structure with alcohol dehydrogenases, and hence both are grouped into a superfamily of medium-chain dehydrogenases and reductases [17]. ZCr homologs, in contrast to alcohol dehydrogenases, lack the Zn-binding domain. In this study, we biochemically characterized A. thaliana P1-ZCr, using recombinant P1-ZCr that had been expressed in Escherichia coli. P1-ZCr showed NADPH:quinone reductase activity, as does the mammalian variety, and also catalyzed the reduction of diamide and its derivative azodicarbonyls, a novel enzyme activity. The substrate specificity and enzymic characteristics of P1-ZCr are compared with those of ZCr homologs, and the implication of the activity of P1-ZCr in protecting cells against oxidative stress is discussed.
M AT E R I A L S A N D M E T H O D S Expression of P1 gene in E. coli E. coli BL21 was used for the expression of P1-ZCr protein. The coding region of the P1 cDNA was amplified by Pfu DNA polymerase (Stratagene, La Jolla, CA, USA) using the P1 cDNA clone [9] as a template with the primers: 5 0 -AACATATGAAATGTACTATACCAGAA-3 0 and 5 0 -ACATATGCTATTGTGGTATTTCAAGAAC-3 0 . After subcloning the amplified fragment into the EcoRV site of the pBluescriptKS(1) (Stratagene), the coding region of the P1 cDNA was excised as an NdeI fragment and subcloned into the NdeI site of the bacterial expression vector pET19b using a T7 promoter (Novagen, Madison, WI, USA), resulting in pETP1. In the pETP1 coding region of the P1 cDNA, the 5 0 -end was fused in-frame with a sequence coding for 10 His residues and an enterokinase site. Expression of the poly(His)-tagged P1-ZCr was induced by adding isopropyl thio-b-d-galactoside, at a final concentration of 0.3 mm, to 0.5 L of a logarithmic culture of the E. coli BL21 [pETP1] grown in Luria±Bertani broth at 20 8C. After 7-h induction, cells were collected, washed with, and then suspended in, 50 mm Tris/HCl, pH 7.8, and frozen at 220 8C until use. Purification of recombinant P1-ZCr The frozen cells were thawed and suspended in extraction medium containing 50 mm Tris/HCl, pH 7.8, 1 mm phenylmethanesulfonyl fluoride, 1.3 mg´mL21 aprotinin, 0.67 mg´mL21 leupeptin and 0.67 mm pepstatin A, then disrupted with a French-pressure cell (49 MPa). After brief ultrasonic treatment of the cell extract to fragment the DNA, the suspension was centrifuged and the resulting supernatant was loaded onto a nickel-chelating Sepharose CL-4B column (Pharmacia Biotech, Tokyo, Japan), which had been prepared according to a laboratory manual [20], and equilibrated with extraction medium containing 0.5 m NaCl (high-salt medium). After
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washing with the high-salt medium, the adsorbed protein was eluted by a linear gradient of imidazole (0±0.5 m) in the highsalt medium made into two bed volumes. The major protein fraction eluted after 0.4 m imidazole was pooled, concentrated by ultrafiltration through a PM 10 membrane (Amicon, Beverly, MA, USA) and further purified by gel filtration with Superdex 200 (Pharmacia), which had been equilibrated with 0.2 m NaCl, 50 mm Tris/HCl, pH 7.8, 1 mm EDTA and 1 mm phenylmethanesulfonyl fluoride. To remove the poly(His) tag, recombinant P1-ZCr (1 mg´mL21) was incubated with enterokinase (0.1 mg´mL21) in 50 mm Tris/HCl, pH 7.5, at 37 8C for 48 h. The purified protein, before and after enterokinase treatment, showed apparent molecular masses of 39.9 kDa and 38.7 kDa, respectively, on SDS/PAGE, which correspond to the values calculated from the amino-acid composition as deduced from the nucleotide sequence of the recombinant P1 gene in the expression vector. Purification of the guinea-pig ZCr Guinea-pig eye lenses were kindly provided by K. Rokutan (Tokushima University Medical School). ZCr was purified from the lens as reported previously [13], with slight modification; we used CM±Sepharose (Pharmacia) and 10 mm potassium phosphate, pH 7.2 after the first Blue Sepharose (Pharmacia) chromatography, rather than the CM-gel and 10 mm Tris/HCl, pH 7.2. Fractions showing NADPH-quinone reductase after CM-Sepharose chromatography were collected, concentrated by ultrafiltration through a PM 30 membrane (Amicon) and subjected to gel filtration using a Superdex 200 column equilibrated with 10 mm Tris/HCl, pH 7.8 and 150 mm NaCl. The final preparation had a molecular mass of 35 kDa on SDS/PAGE, consistent with that reported previously [13]. Determination of P1-ZCr concentration The concentration of purified P1-ZCr was determined from the absorbance at 280 nm, based on an absorption coefficient of 81.2 mm21´cm21, which was calculated from the deduced amino-acid composition of a dimer, as P1-ZCr is a dimer (described later), using the empirically determined coefficients of Trp and Tyr residues [21]. Determination of molecular mass of P1-ZCr by SDS/PAGE and gel filtration Marker proteins and their molecular masses (in kDa) included phospholipase A (94), albumin (67), ovalbumin (43), carbonic anhydrase (30), trypsin inhibitor (20.1) and a-lactalbumin (14.4) for SDS/PAGE (Nippon Bio-Rad Lab., Osaka, Japan); and ferritin (440), catalase (232), aldolase (158), albumin (67), ovalbumin (43) and chymotrypsinogen A (25) for gel filtration (Pharmacia). Gel filtration was performed through a Superdex 200 HR10/30 column (Pharmacia) equilibrated with 50 mm Tris/HCl, pH 7.8, and 200 mm. A 200-mL solution of P1-ZCr at 1 mg´mL21 in 50 mm Tris/HCI, pH 7.8, was injected into the column, and eluted with the equilibrating medium at a flow rate of 0.2 mL´min21. Determination of enzyme activities The standard reaction mixture contained 50 mm Tris/HCl, pH 7.5; 50 mm NADPH, an electron acceptor at the indicated concentration and 12.3 nm P1-ZCr. The reaction was started by addition of the enzyme, and the initial oxidation rate (3±15 s) of NADPH was determined by a decrease in
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Diamide reductase activity in plant z-crystallin (Eur. J. Biochem. 267) 3663
absorbance at 340 nm, using an absorption coefficient of 6.2 mm21´cm21. Because diamide and 1,1 0 -(azodicarbonyl) dipiperidine (dipiperidine) showed significant absorbance at 340 nm with absorption coefficients of 1.2 and 3.7 mm21´cm21, respectively, and were bleached upon reduction, the oxidation rate of NADPH was corrected for the reduction of the electron acceptors, using the stoichiometry NADPH : electron acceptor 1 : 1, as determined later. Reduction of ferricytochrome c was followed by the absorbance increase at 550 nm, using an absorption coefficient of 29 mm21´cm21. The detergent deoxy-BIGCHAP (Dojindo Lab., Kumamoto, Japan) was added at 1.4 mm to the reaction mixture for solubilizing phylloquinone and decyl-plastoquinone (DPQ). At this concentration the detergent did not affect the enzyme activity of the other quinones and diamide. All the activity was corrected for the nonenzymic oxidation rate of NADPH by the electron acceptors, which was determined before the addition of P1-ZCr. Analytical methods Analytical TLC was performed on a silica-gel plate (Merck 5715, 0.25 mm). Elemental analyses were performed on a Yanaco MT-5. 1H NMR and 13C NMR were recorded on a Varian VXR-200 (200 MHz) with tetramethylsilane as an internal standard in CDCl3. Infrared spectra were recorded on a Hitachi U-215 spectrophotometer, and mass spectra on a JEOL JMS700 spectrometer. Synthesis of 1,2-bis(N,N-dimethylcarbamoyl)hydrazine 1,2-Bis(N,N-dimethylcarbamoyl)hydrazine (hydrazodicarbonamide) was synthesized from hydrazine hydrate and N,Ndimethylcarbamoyl chloride as reported previously [22].
Recrystallization from hot CHCl3-hexane afforded a compound appearing as colorless needles, m.p. 219.2±219.5 8C (corrected); RF (chloroform/ethanol/acetic acid 90 : 10 : 5) 0.46; IR (KBr) nmax 3250 (broad), 1620, 1520, 1360, 1180, 860 cm21; 1 H NMR (200 MHz) d 7.17 (broad singlet, 2H, 2 NH) and 2.96 (singlet, 12H, 4 CH3); 13C NMR d 159.3 (CO) and 36.1 (CH3). Elemental analysis of the obtained compound found C, 41.21; H, 8.07, N 32.29%, while the calculated values for C6H14N4O2 were C, 41.37; H, 8.10; N, 32.16%. MS (electron impact) gave fragment peaks of m/z 174 (M1, 32%), 129 (53), 72 (100). High resolution mass spectrometry (HRMS) for M1 found 174.1113, while the calculated value was 174.1117.
Chemicals and enzymes Azodicarbonamide, N,N-dimethylcarbamoyl chloride and hydrazine hydrate were obtained from Nakalai Tesque (Kyoto, Japan). Catalase from bovine liver, DPQ, diamide, menaquinone and trans-2-butenoyl CoA were from Sigma Japan (Tokyo). Dipiperidine, methyl red and 9,10-phenanthrenequinone (PAQ) were from Tokyo Chemical Industry Co., Ltd. (Tokyo). Enterokinase was from Boehringer Mannheim (Tokyo). Ferricytochrome c from horse heart, Mn-superoxide dismutase (SOD) from Bacillus sp. and phylloquinone were from Wako Pure Chemicals (Tokyo). Ferredoxin was prepared from spinach leaves as reported previously [23]. Malondialdehyde was prepared by hydrolyzing 1,1,3,3-tetraethoxypropane [24]. Monodehydroascorbate was produced in situ by oxidizing 2.5 mm ascorbic acid with ascorbate oxidase (Toyobo Co., Ltd, Tokyo) to give a steady-state concentration of 11 mm, which was determined by the absorbance at 360 nm, using an absorption coefficient of 3.3 mm21´cm21 [25].
Table 1.Comparison of substrate specificity of Arabidopsis P1-ZCr and guinea-pig ZCr. Activity was determined as described in Materials and methods. Activity was corrected for nonenzymic oxidation of NADPH, if any, by an electron acceptor. The kcat values of the electron acceptors of which Km values were not determined were estimated from the maximum velocities, which were determined at the parenthesized concentrations of the acceptors, where reaction velocities were saturated. Guinea-pig Zcra
Arabidopsis P1-ZCr Electron acceptor
kcat (s21)
Km (mm)
kcat /Km
kcat (s21)
Km (mm)
kcat /Km
9,10-Phenanthrenequinone (PAQ) 5-Hydroxy-1,4-naphtoquinone ( juglone) 1,4-Benzoquinone 1,2-Naphthoquinone
98 1.9 12 54 (25 mm)
0.65 11 152 NDb
151 0.17 0.079 ±
19 4.9 5.9 39 (100 mm)
13 27 143 ±
1.5 0.18 0.04 ±
2,6-Dichlorophenolindophenol
7.5 (30 mm)
ND
±
1.5 (100 mm)
±
±
5-Hydroxy-2-methyl-1,4-naphtoquinone (plumbagin)
3.8 (100 mm)
ND
±
0.37 (250 mm)
±
±
1,4-Naphthoquinone
3.0 (25 mm)
ND
±
1.0 (250 mm)
±
±
Phylloquinone (vitamin K1) Menaquinone (vitamin K2) Menadione (vitamin K3) Decyl-plastoquinone (DPQ) Ferricytochrome c
0c 0c 0c 0.10 0.03
± ± ± 25 20
± ± ± 0.004 0.002
± ± 0 ± 0d
± ± ± ± ±
± ± ± ± ±
a
Recalculated from [11].
b
Not determined. c Determined at 100 mm of the electron acceptor.
d
Reference [10].
3664 J. Mano et al. (Eur. J. Biochem. 267)
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Table 2. Specificity of P1-ZCr to azo compounds and analogs. I, azodicarboxylic acid bis[dimethylamide]; diamide; II, 1,1 0 -(azocarbonyl)dipeperidine; dipiperidine; III, azocarbonamide; IV, azocarboxylic acid dimethyl ester; V, 4-dimethylamino-azobenzene; methyl yellow; VI, 4-dimethylaminoazobenzene-2 0 -carboxylic acid; methyl red; VII, dimethylmaleate (cis-form) and diethylfumarate (trans-form); VIII, trans2-butenoyl-CoA (crotonoyl-CoA).
k cat values for the o-quinones PAQ and 1,2-naphtoquinone than those for p-quinones. Futhermore, P1-ZCr was incapable of reducing 100 mm menadione, 1.5 mm ferricyanide and 50 mm methylviologen like guinea-pig ZCr [13,14]. Preference for o-quinones over p-quinones, and the inability to recognize menadione and ferricyanide as substrates, clearly distinguished P1-ZCr and guinea-pig ZCr from the flavin-containing NAD(P)H-quinone oxidoreductases (DT-diaphorases; EC 1.6.99.2) in plants [29,30] and animals [31]. In an attempt to identify the physiological substrates of P1-ZCr, we tested several electron acceptor candidates which either occur naturally or are related to oxidative stress. P1-ZCr did not catalyze the reduction of phylloquinone (vitamin K1), a natural quinone in plants. Slight activity was observed for DPQ, an analog of the naturally occurring plastoquinone, with a Km value of 25 mm. Unlike guinea-pig ZCr, Arabidopsis P1-ZCr recognized ferricytochrome c from horse heart as the electron acceptor with a Km value of 20 mm. The molecular activity and specificity of P1-ZCr for these two electron donors were, however, very low compared with those for other quinones (Table 1). Presumably these activities with DPQ and cytochrome c were physiologically irrelevant, because P1-ZCr is most probably localized in the cytosol [9]. The following were incompatible as electron acceptors with 0.5 mm NADPH in 50 mm Tris/HCl at pH 7.5 using 5 nm P1-ZCr: 260 mm dioxygen, 9 mm ferredoxin from spinach, 0.2 mm oxidized glutathione (GSSG), 1 mm H2O2, 40 mm tert-butylhydroperoxide, 50 mm cumene hydroperoxide, 10 mm l-methionine sulfoxide, 5 mm malondialdehyde and 11 mm monodehydroascorbate. Thus, P1-ZCr is distinguished from NADPH oxidase, ferredoxin-NADP1 reductase, glutathione reductase, NADPH peroxidase, methionine sulfoxide reductase, monodehydroascorbate reductase and DT-diaphorase.
a
Diamide and its derivatives are the electron acceptors of P1-ZCr
Enzymic reaction could not be estimated because of very fast nonenzymic oxidation of NADPH by IV.
R E S U LT S A N D D I S C U S S I O N Arabidopsis P1-ZCr is a dimer A molecular mass of 38.7 kDa was determined for the enterokinase-treated P1-ZCr on SDS/PAGE under both reducing and nonreducing conditions. This corresponded well with the monomer size calculated from the amino-acid sequence (38 284 Da). On gel filtration, the molecular mass was determined to be 83.2 kDa. Thus, Arabidopsis P1-ZCr is a noncovalent homodimer, as is the quinone oxidoreductase (QOR) in E. coli [26], a ZCr homolog. This contrasts with the ZCrs in mammalian lenses, which are tetramers [27,28]. The P1-ZCr comprising the N-terminal poly(His) tag also formed a dimer. Results described hereafter were obtained using the P1-ZCr comprising the poly(His) tag and are shown on the basis of the molecular mass of the dimer.
NADPH:quinone oxidoreductase activity of P1-ZCr The NADPH:quinone oxidoreductase activity reported for ZCrs from guinea pig [13,14] and camel [15] was also observed for Arabidopsis P1-ZCr (Table 1). The substrate specificity of P1-ZCr was very similar to that of guinea-pig ZCr; both are more specific for PAQ than for juglone and 1,4-benzoquinone, as judged by the k cat /Km values. Both enzymes showed higher
We found that P1-ZCr catalyzes the reduction of diamide by NADPH (Table 2, I). In 50 mm NADPH, the kcat value for diamide was comparable with those for quinones. Two other azodicarbonyls, dipiperidine (II) and azodicarbonamide (III), were also found to serve as electron acceptors, with the highest specificity for III. This ability to reduce azodicarbonyls is not due to the N-terminal poly(His) tag in the recombinant P1-ZCr, because the same activity was observed for the tag-free P1-ZCr. Methyl yellow (V) and methyl red (VI) did not serve as electron acceptors for P1-ZCr, distinguishing this protein from the flavin-containing azoreductases in human liver [32], including NADPH-cytochrome c reductase [33,34]. Neither diethylmaleate (VII, cis-form), a glutathione-reactive compound, nor its trans-isomer diethylfumarate was a substrate of P1-ZCr. Because of the limited availability of azodicarbonyls, we could not judge whether the amino nitrogen atoms on compounds I±III are necessary for substrate recognition. To our knowledge, this is the first report of the enzymic reduction of azodicarbonyls using NAD(P)H. Acyl-CoA : NADP1 trans-2-oxidoreductase (enoyl reductase) activity is detected in several ZCr homologs, specifically, in Streptomyces collinus crotonyl-CoA reductase [35] and in the ZCr-homologous domains of the rat fatty acid synthase [36,37] and Saccharopolyspora eythrea erythonolide synthase [38]. In the crotonoyl-CoA reductase reaction, the C±C double bond in trans-2-butenoyl CoA (VIII) is divalently reduced to the saturated butyryl-CoA. This resembles the divalent reduction of the azo bond in the P1-ZCr-catalyzed diamide reduction
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Diamide reductase activity in plant z-crystallin (Eur. J. Biochem. 267) 3665
(described later). However, P1-ZCr did not catalyze the reduction of trans-2-butenoyl CoA, and hence is distinct from enoyl-CoA reductase. The close similarity between the quinone specificity of P1-ZCr and guinea-pig ZCr tempted us to anticipate diamide reductase activity in guinea-pig ZCr as well. However, purified guinea-pig ZCr retaining the reported quinone reductase activity [14] did not show diamide-reducing activity (data not shown). These results indicate that P1-ZCr is a new class of NADPH oxidoreductase in the ZCr family. Distinct from guinea-pig ZCr, Arabidopsis P1-ZCr showed specificity to dual classes of electron acceptors, and hence we can refer to it as NADPH:azodicarbonyl/quinone reductase. Our results further demonstrate the diversity of the enzymic characteristics of ZCr homologs, which is likely reflected in the diverse physiological functions of the proteins in this family. The P1 gene confers tolerance to diamide in yap1D yeast [9], which is attributable to the diamide-reducing activity of the P1-ZCr. Radical chain reaction accompanying the univalent reduction of quinones Quinone reductase catalyzes either univalent or divalent reduction of quinones (Q) primarily to produce semiquinones (QH´) or quinols (QH2), respectively. Production of QH´ by univalent catalysts is detectable by the reduction of ferricytochrome c by the QH´ to ferrocytochrome c; QH´ 1 ferricytochrome c ! Q 1 ferrocytochrome c
1
whereas in the reaction of divalently reducing enzymes, such as DT diaphorase, no reduction of ferricytochrome c is detectable [39]. That guinea-pig ZCr is a univalent catalyst was demonstrated by this method and by detection of semiquinone radicals with ESR [14]. Like guinea-pig ZCr, P1-ZCr showed the quinone-dependent catalysis of ferricytochrome c reduction (Fig. 1). Ferricytochrome c itself was a very poor electron acceptor for P1-ZCr (Table 1; Fig. 1, trace A). The addition of PAQ accelerated the reduction of ferricytochrome c (trace B). In this case, the reductant for ferricytochrome c could be QH´
Fig. 1. Reduction of ferricytochrome c by semiquinones of PAQ and O2 2 . Absorbance changes at 550 nm of the reaction mixture containing 50 mm Tris/HCl, pH 7.5, 50 mm NADPH and 12 mm ferricytochrome c were monitored (A). P1-ZCr was added to 25 nm where indicated. PAQ at 5 mm (B) and SOD at the indicated concentrations (C, D) were added where indicated. Addition of SOD . 75 U´mL21 did not decrease the rate of ferricytochrome c reduction further (data not shown).
´ as well as O2 2 , which is produced via the auto-oxidation of QH as follows:
1 QH´ 1 O2 ! Q 1 O2 2 1H
O2 2 1 ferricytochrome c ! O2 1 ferrocytochrome c
2
3
Reduction of ferricytochrome c (reaction 3) was partly suppressed by the addition of 30 U´mL21 SOD (trace C), but not eliminated totally by even 75 U´mL21 SOD (trace D) or more (data not shown). This SOD-insensitive part was ascribable to QH´ (reaction 1). Thus, P1-ZCr is a univalent catalyst, as is guinea-pig ZCr, and primarily produces QH´. At 40 mm ferricytochrome c, at which the reduction of ferricytochrome c becomes saturated, the rate of SODinsensitive ferricytochrome c reduction was 3.0 mm´min21, which was almost twice the NADPH oxidation rate, 1.64 mm´min21, as determined separately under the same conditions. In this case, 91% of the QH´ produced reduced ferricytochrome c directly (reaction 1), and the rest reduced O2 to O2 2 (reaction 2). The generation of O2 2 in a univalent quinone reductase system leads to a radical chain reaction [40]. In the case of the P1-ZCr reaction, the following reaction sequences are likely to occur. QH´, produced via the univalent reduction of Q in the P1-ZCr reaction (4), is either spontaneously disproportionated to Q and QH2 (5), or interacts with O2 to produce O2 2 (2). NADPH 1 2Q 1 H1 ! NADP1 1 2QH´
4
2QH´ ! Q 1 QH2
5
O2 2
reacts with the quinol to produce the semiquinone, and thereby propagates the chain reaction [40]: 1 ´ O2 2 1 QH2 1 H ! QH 1 H2 O2 :
6
The chain reaction is terminated by the disproportionation of O2 2: 1 2O2 2 1 2H ! H2 O2 1 O2 :
O2 2
7
When the spontaneous disproportionation of (reaction 7) is negligible compared with the oxidation of QH2 by O2 2 (reaction 6), the consequence of the chain reaction (reactions 2, 4±6) is
3666 J. Mano et al. (Eur. J. Biochem. 267)
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Fig. 2. P1-ZCr-catalyzed oxidation of NADPH in the presence of PAQ under aerobic conditions. Absorbance changes at 340 nm of the reaction mixture containing 50 mm Tris/HCl, pH 7.5, 12.5 nm P1-ZCr and 50 mm NADPH were monitored. Down arrow, PAQ added to the reaction mixture to give 3 mm. Upper trace, P1-ZCr omitted. Middle trace, 75 U´mL21 SOD included. Inset, enlarged traces of the initial phase of the P1-ZCr reaction at various SOD concentrations.
the oxidation of NADPH by O2 to produce H2O2, without any consumption of Q, such that NADPH 1 O2 1 H1 ! NADP1 1 H2 O2 ;
8
sum of Eqns (4), (5), (2) and (6). In contrast, when SOD is added the consumption of NADPH would be equimolar to the added Q, because the chain reaction is quickly terminated by the rapid disproportionation of O2 2 (reaction 7), such that NADPH 1 Q ! NADP1 1 QH2 : For P1-ZCr with PAQ as the electron acceptor under aerobic conditions, NADPH was oxidized in an excess amount compared with that for the quinone (Fig. 2, lower trace). After all the NADPH had been oxidized, PAQ was not reduced at all, as judged by its absorbance at 430 nm (data not shown). Concomitantly, O2 was consumed equimolar to the oxidized NADPH, and by the subsequent addition of catalase almost half-equimolar O2 to the consumed O2 was evolved (Fig. 3, trace A). Thus, H2O2 accumulated via spontaneous disproportionation of O22, corresponding with the situation represented by reaction 8. In the absence of O2, NADPH was oxidized
equimolar to the added quinone (data not shown), as predicted above. When SOD was added, the chain reaction was terminated due to the suppression of reaction 6; NADPH oxidation became almost equimolar to the added PAQ (Fig. 2, middle trace), and O2 consumption was largely suppressed (Fig. 3, trace B). It should be noted that the initial rate of NADPH oxidation was not affected by SOD (Fig. 2, inset), indicating that reaction 4 limits the rate of the overall reaction in either case. Therefore, the initial rates of NADPH-oxidation by the respective electron acceptors, determined even in the absence of SOD, represent the intrinsic reaction rate by P1-ZCr. Divalent reduction of diamide, without producing O2 2 In contrast to the quinone reduction, P1-ZCr catalyzed the reduction of diamide by equimolar NADPH even in the absence of SOD (Fig. 4). This 1 : 1 stoichiometry was not affected by deoxygenation or by SOD, and neither oxygen consumption nor ferricytochrome c reduction was observed during the reaction (data not shown). The 1 : 1 stoichimetry of (added electron acceptor) vs. (oxidized NADPH) was also confirmed for
Fig. 3. Oxygen uptake during the P1-ZCr-catalyzed oxidation of NADPH in the presence of PAQ. To a reaction mixture containing 100 mm NADPH in 50 mm Tris/HCl, pH 7.5, were added 20 mm PAQ and 25 nm P1-ZCr as indicated (A). (B) SOD 150 U´mL21 was included in the reaction mixture. Catalase, 580 U´mL21, was added where indicated.
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Diamide reductase activity in plant z-crystallin (Eur. J. Biochem. 267) 3667
Inhibitors of quinone and diamide reduction
Fig. 4. P1-ZCr-induced oxidation of NADPH by diamide under aerobic conditions. Diamide of the indicated amount was sucessively added to a reaction mixture (1 mL) containing 50 mm Mes/NaOH, pH 6.5, 100 mm NADPH and 25 nm P1-ZCr.
dipiperidine (II) and azodicarbonamide (III) (data not shown). Thus, the reduction of azodicarbonyls is not accompanied by either production of O2 2 or the radical chain reaction. The reduced product of diamide was expected to be the hydrazodicarbonamide (IX; Scheme 1), because it is produced by the nonenzymic divalent reduction of diamide by glutathione [41]. The product of diamide reduction in the P1-ZCr reaction was identified as follows. The reaction mixture (0.58 L) containing 116 mmol diamide, 116 mmol NADPH and 5 nmol of P1-ZCr in 10 mm ammonium carbonate, pH 6.5, was incubated at 25 8C for 10 min to complete the reaction. The mixture was lyophilized and extracted with chloroform. Recrystallization afforded colorless crystals, which showed RF 0.46; IR (KBr) nmax 3250 (br), 1620, 1520 (br), 1360, 1180, 860 cm21; 1H NMR (200 MHz) d 7.00 (broad singlet, 2H, 2 NH) and 2.97 (singlet, 12H, 4 CH3); 13C NMR d 159.2 (CO) and 36.1 (CH3); MS (electron impact) m/z 174 (M1, 38), 129 (44), 72 (100); and HRMS for M1 found 174.1115. Comparing these values with those of the synthetic authentic compound (see Materials and methods), the reaction product was identified as hydrazodicarbonamide (IX). Thus, P1-ZCr catalyzes the reaction in Scheme 1. The divalent reduction of diamide by P1-ZCr contrasts with the tetravalent reductive fission of azo compounds catalyzed by the flavin-containing azo reductases [33,42]. We concluded that P1-ZCr exhibits dual modes of reaction; univalent reduction of quinones and divalent reduction of azodicarbonyls.
Scheme 1. The enzymic reduction of diamide by P1-ZCr.
Dicumarol and nitrofurantoin are competitive inhibitors of guinea-pig ZCr against electron acceptors, but are uncompetitive against NADPH [14]. The actions of these inhibitors on Arabidopsis P1-ZCr were different to those on guinea-pig ZCr. Dicumarol showed a mixed-type inhibition against both the electron donor NADPH and the acceptors diamide and PAQ (Fig. 5). Inhibition against NADPH in the diamide-reducing reaction was near-noncompetitive (Fig. 5D), indicating that the binding of NADPH does not strongly affect that of dicumarol. This action of dicumarol on P1-ZCr is distinct from that on flavin-containing quinone reductases, in which this inhibitor is competitive against NAD(P)H but independent of the electron acceptor [43]. Nitrofurantoin showed more complex features (Fig. 6); it was noncompetitive against PAQ (Fig. 6A) and uncompetitive against NADPH in PAQ-reducing reaction (Fig. 6B). In contrast, the inhibition induced by nitrofurantoin was a mixed-type against diamide (Fig. 6C) and noncompetitive against NADPH in the diamide-reducing reaction (Fig. 6D). These differences in the inhibitor actions between quinone-reducing and diamide-reducing reactions could be a reflection of the difference in the binding of the two substrates to the enzyme. The quinone-binding site in E. coli QOR is thought to be located in a cleft formed by helices a1 and a2 and strand b1, and the amino-acid residues lining the substrate-binding sites are conserved between QOR and the guinea-pig ZCr [44]. Such amino-acid residues are not found in P1-ZCr, although P1-ZCr showed a substrate specificity to quinones similar to that in guinea-pig ZCr. This structural difference would result in the characteristic reactivity of P1-ZCr to azodicarbonyls and the different actions of inhibitors between guinea-pig ZCr and Arabidopsis P1-ZCr. This raises an intriguing question; namely, which tertiary structure of the substrate binding site of P1-ZCr ensures the dual substrate specificity? Nucleotide specificity The ZCr and alcohol dehydrogenase families have similar nucleotide-binding sequences [17], but the former appears to prefer NADPH to NADH, while the latter does not. Guinea-pig ZCr [14] and crotonoyl-CoA reductase [35] are highly specific for NADPH. E. coli QOR also exhibits a preference for NADPH compared with NADH (N. E. Dixon, personal communication). Based on a comparison of the three-dimensional structures of E. coli QOR and lactate dehydrogenase, Edwards et al. [44] suggested that in the G (or A) XXGXXG motif of the nucleotide-binding regions of ZCr homologs, the Ala residue next to the first Gly (or Ala) allows the accommodation of the adenine ribose phosphate group, while alcohol dehydrogenases lack this Ala in their GXGXXG motif. P1-ZCr also has an Ala after the first Gly in the GXXGXXG motif at its nucleotidebinding site (Fig. 7). As expected from this sequence, P1-ZCr showed a higher preference for NADPH than NADH. In the
3668 J. Mano et al. (Eur. J. Biochem. 267)
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Fig. 5. Inhibition of the PAQ-reducing activity (A, B) and diamide-reducing activity (C, D) of P1-ZCr by dicumarol. PAQ-reducing activities were determined in 50 mm Tris/HCl, pH 7.5 and diamide-reducing activities were determined in 50 mm Mes/NaOH, pH 6.5. Reaction velocities are expressed in arbitrary units. Km and Vmax values were determined by nonlinear regression of the data using the Michaelis±Menten equation with the curve-fitting program built into the software kaleidagraph (Synergy Software, Reading, PA). Each data point is an average of two runs.
PAQ-reducing reaction, the specificity for NADPH, as determined by kcat/Km, was 1.7 107, while that for NADH was 1.2 104 ; i.e. a 1000-fold difference. In the diamide-reducing reaction, kcat/Km for NADPH was 1.6 107, while the activity for NADH was so low that we were not able to estimate kcat and Km values accurately, indicating more strict selectivity for NADPH. The Km values for NADPH in the PAQ-reduction and diamide-reduction were 2.5 mm (at 5 mm PAQ) and 8.2 mm (average at 2±120 mm diamide), respectively. Thiol modifiers: participation of a Cys residue in the binding of NADPH Both PAQ-reducing and diamide-reducing activities were irreversibly inhibited by N-ethylmaleimide with equal sensitivity
(Fig. 8). The activities were also sensitive to p-chloromercuribenzoic acid, indicating that a thiol group(s) participates in the catalytic activities, as reported for guinea-pig ZCr [14]. Inhibition of the reductase activities of P1-ZCr by thiolmodifiers was suppressed by NADPH and NADP1, but not by NADH or diamide (Table 3). This indicates the participation of at least one Cys residue at the NADPH binding site. P1-ZCr has five Cys residues, Cys50, Cys151, Cys181, Cys211 and Cys254. Cys151 and Cys181 reside in or near the NADPHbinding site (Fig. 7, asterisks). The quinone-reducing activity of guinea-pig ZCr was also sensitive to thiol modifiers [14]. However, none of the Cys residues are conserved between guinea-pig ZCr and Arabidopsis P1-ZCr, indicating that the respective roles of the modified Cys residues in these two proteins are distinct.
Fig. 6. Inhibition of the PAQ-reducing activity (A, B) and diamide-reducing activity (C, D) of P1-ZCr by nitrofurantoin. Activities were determined as described in Fig. 5.
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Fig. 7. Alignment of amino-acid sequences around the NADPH-binding site in ZCr homologs. Top to bottom: P1-ZCr from A. thaliana (P1) [9], QOR from E. coli (Dixon & Lilley 1992, EMBL databank accession no. P28304), ZCr from guinea pig [57] and crotonoyl-CoA reductase from S. collinus (CCR) [35]. Numbers in parentheses represent the position (count from the N-terminus) of the left-hand residues in the compared sequences. Residues identical in all four proteins are shaded in dark gray, similar residues are shaded in light gray. The NADPH-binding region is underlined. The location of the G (or A) XXGXXG motif characteristic to ZCR homologs is shown below the aligned sequences. Cys residues of P1ZCr in this region are marked by an asterisk (see text).
Effects of pH on quinone-and diamide-reducing activities P1-ZCr showed a sharp pH profile for PAQ-reduction, peaking at pH 8, with half-maximal rates at pH 7.5 and pH 9.2, and there was no substantial reduction of PAQ at pH , 7.0. In contrast, diamide was reduced over a broader pH range, with a maximum rate at pH 6.5±7 and half-maximal rates at pH 5.5 and 8.3 (data not shown). Thus, whether P1-ZCr functions as a quinone reductase or an azodicarbonyl reductase depends on the pH of its microenvironment, as well as on the availability of the substrates. Possible physiological functions Induced expression of the P1 gene by oxidative stress in A. thaliana [9] suggests that Arabidopsis P1-ZCr exerts protective functions against stress. Indeed, overexpression of the P1 gene improved the tolerance of tobacco plants to methyl viologen (E. Belles-Boix, L. Slooten, J. Mano, E. Babiychuk, K. Asada, M. Van Montagu, D. Inze & S. Kushnir, unpublished
Fig. 8. Time course of inactivation of PAQ-reducing and diamidereducing activities of P1-ZCr by N-ethylmaleimide. P1-ZCr at 1.25 mm was incubated in 2 mm N-ethylmaleimide, 50 mm Tris/HCl, pH 7.5 at 25 8C. At the indicated time, 20 mL aliquots of the mixture were transferred into a 1-mL assay mixture of 50 mm Tris/HCl, pH 7.5, 100 mm NADPH containing either 100 mm diamide (X) or 50 mm PAQ (P).
Table 3. Effects of substrates on the p-chloromercuribenzoic acidinduced inactivation of diamide-reducing activity of P1-ZCr. P1-ZCr (0.63 mm) was incubated at 25 8C in 2 mm p-chloromercuribenzoic acid and 50 mm Tris/HCl, pH 7.5, containing the indicated compound. Enzyme activities before and 5 min after the addition of p-chloromercuribenzoic acid were determined in the reaction mixture containing 100 mm NADPH, 100 mm diamide and 50 mm Tris/HCl, pH 7.5. Average percent inactivation in response to the treatment of two runs is shown. Similar effects of NADPH, NADP1, NADH and diamide were observed for the N-ethylmaleimide-induced inactivation (not shown) Added in the preincubation with p-chloromercuribenzoic acid
Inactivation (%)
None NADPH, 1 mm NADH, 1 mm NADP1, 1 mm Diamide, 0.5 mm
89 0 88 26 87
results). The mechanism of the tolerance, however, remains unclear, largely because the physiological electron acceptor(s) for P1-ZCr has not been identified. The reduction of azodicarbonyls by Arabidopsis P1-ZCr, revealed in this study, suggests the detoxification of azo compounds such as diazoate-derivatives by this enzyme if they are converted to azodicarbonyls in plant cells. Dioazoates are strong alkylating intermediates produced via the a-hydroxylation of nitrosamines by cytochrome P450 [45]. Cycad plants accumulate the carcinogenic and cytotoxic azoxy compounds macrozamin and cycasin in their nuts [46]. Similar azoxy compounds are produced by several actinomycetes [47,48] and fungi [49]. Arabidopsis P1-ZCr might protect cells when azoxy compounds are produced endogenously under stressed conditions or introduced exogenously by infection. Alternatively, it is possible that Arabidopsis P1-ZCr functions as a quinone reductase. Possible substrate quinones are not abundant in the cytosol, but under severe stress the quinones might be liberated from the cell compartments where they are normally sequestered, as exemplified by the release of polyphenols from vacuoles and polyphenol oxidase from thylakoid lumen upon the disruption of cells. Quinone reduction would not lead to the radical chain reaction, because SOD is ubiquitous in cells. Thus, the quinones are detoxified through the conjugation of the quinols with UDP-glucuronate [40]. If the produced O2 2 is not scavenged effectively, the radical chain reaction starts, and H2O2 accumulates. As the
3670 J. Mano et al. (Eur. J. Biochem. 267)
endogenously produced H2O2 induces a battery of defense genes, e.g. glutathione S-transferase [50], PR-1 [51] and ascorbate peroxidase [52,53], P1-ZCr would enforce cellular defense by providing O2 2 and H2O2 via its quinone-reducing reaction. TED2, a ZCr-homologous gene in plants, is expressed at an early stage of the development of tracheary elements in Zinnia elegance [18], and has been proposed to function in programmed cell death via redox-mediation or propagation of oxidative stress [54]. A similar function has been suggested for a human ZCr homolog [55]. Another homologous gene, YML131w, in S. cerevisiae is regulated by yAP1 [56], specifically activated under oxidative stress. These gene products are also likely to have oxidoreductase activity in common. However, detailed biochemical characterization is required to elucidate the function of each protein, because the ZCr homologs show diverse activities, as exemplified by this work for Arabidopsis P1-ZCr.
ACKNOWLEDGEMENTS This study was supported by a Grant-in-Aid for International Cooperative Research, and Grants-in-Aid for Scientific Research (no. 09044219 and no. 09760116) from the Ministry of Education, Science and Culture, Japan, and also by a grant from the Human Frontier Science Program. The authors would like to express their thanks to Professor L. Slooten for stimulating discussion, and Professor N. E. Dixon for kindly providing unpublished data.
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