Arabidopsis CYP735A1 and CYP735A2 Encode Cytokinin Hydroxylases That Catalyze the Biosynthesis of trans-Zeatin
2004; Elsevier BV; Volume: 279; Issue: 40 Linguagem: Inglês
10.1074/jbc.m406337200
ISSN1083-351X
AutoresKentaro Takei, Tomoyuki Yamaya, Hitoshi Sakakibara,
Tópico(s)Plant Molecular Biology Research
ResumoCytokinins (CKs), a group of phytohormones, are adenine derivatives that carry either an isoprene-derived or an aromatic side chain at the N6 terminus. trans-Zeatin (tZ), an isoprenoid CK, is assumed to play a central physiological role because of its general occurrence and high activity in bioassays. Although hydroxylation of isopentenyladenine-type CKs is a key step of tZ biosynthesis, the catalyzing enzyme has not been characterized yet. Here we demonstrate that CYP735A1 and CYP735A2 are cytochrome P450 monooxygenases (P450s) that catalyze the biosynthesis of tZ. We identified the genes from Arabidopsis using an adenosine phosphate-isopentenyltransferase (AtIPT4)/P450 co-expression system in yeast. Co-expression of AtIPT4 and CYP735A enabled yeast to excrete tZ and the nucleosides to the culture medium. In vitro, both CYP735As preferentially utilized isopentenyladenine nucleotides rather than the nucleoside and free base forms and produced tZ nucleotides but not the cis-isomer. The expression of CYP735A1 and CYP735A2 was differentially regulated in terms of organ specificity and response to CK. Root-specific induction of CYP735A2 expression by CK suggests that the trans-hydroxylation is involved in the regulation of CK metabolism and signaling in roots. Cytokinins (CKs), a group of phytohormones, are adenine derivatives that carry either an isoprene-derived or an aromatic side chain at the N6 terminus. trans-Zeatin (tZ), an isoprenoid CK, is assumed to play a central physiological role because of its general occurrence and high activity in bioassays. Although hydroxylation of isopentenyladenine-type CKs is a key step of tZ biosynthesis, the catalyzing enzyme has not been characterized yet. Here we demonstrate that CYP735A1 and CYP735A2 are cytochrome P450 monooxygenases (P450s) that catalyze the biosynthesis of tZ. We identified the genes from Arabidopsis using an adenosine phosphate-isopentenyltransferase (AtIPT4)/P450 co-expression system in yeast. Co-expression of AtIPT4 and CYP735A enabled yeast to excrete tZ and the nucleosides to the culture medium. In vitro, both CYP735As preferentially utilized isopentenyladenine nucleotides rather than the nucleoside and free base forms and produced tZ nucleotides but not the cis-isomer. The expression of CYP735A1 and CYP735A2 was differentially regulated in terms of organ specificity and response to CK. Root-specific induction of CYP735A2 expression by CK suggests that the trans-hydroxylation is involved in the regulation of CK metabolism and signaling in roots. Cytokinins (CKs), 1The abbreviations used are: CK, cytokinin; CKX, cytokinin oxidase/dehydrogenase; cZ, cis-zeatin; DZ, dihydrozeatin; DZR, dihydrozeatin riboside; HMBDP, 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate; iP, isopentenyladenine; iPR, isopentenyladenine riboside; iPRDP, isopentenyladenine riboside 5′-diphosphate; iPRMP, isopentenyladenine riboside 5′-monophosphate; iPRTP, isopentenyladenine riboside 5′-triphosphate; IPT, adenosine phosphate-isopentenyltransferase; LC-MS, liquid chromatography-mass spectrometry; MEP, methylerythritol phosphate; MVA, mevalonate; P450, cytochrome P450 monooxygenase; tZ, trans-zeatin; tZR, trans-zeatin riboside; tZRMP, trans-zeatin riboside 5′-monophosphate; CHES, 2-(cyclohexylamino)ethanesulfonic acid; HPLC, high pressure liquid chromatography. a group of phytohormones, are involved in the regulation of various processes in plant growth and development such as cell division, leaf senescence, and nutritional signaling (1Mok M.C. Mok D.W.S. Mok M.C. Cytokinins: Chemistry, Activity, and Function. CRC Press, Inc., Boca Raton, FL1994: 155-166Google Scholar). The major natural CKs are adenine derivatives that carry an isoprene-derived side chain at the N6 terminus, although CKs with an aromatic side chain occur in some plant species (2Strnad M. Physiol. Plant. 1997; 101: 674-688Crossref Google Scholar). Small side chain variations that exist in isoprenoid CKs include the absence or presence of a hydroxyl group at the end of the prenyl side chain and the stereoisomeric position (3Mok D.W. Mok M.C. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001; 52: 89-118Crossref PubMed Scopus (894) Google Scholar). Common derivatives are 6-(3,3-dimethylallylamino)purine, 6-((E)-4-hydroxy-3-methylbut-2-enylamino)purine, and 6-((Z)-4-hydroxy-3-methylbut-2-enylamino)purine, whose trivial names are isopentenyladenine (iP), trans-zeatin (tZ), and cis-zeatin (cZ), respectively (see Fig. 1). The reduction of the double bond in the tZ side chain, which is catalyzed by a zeatin reductase, forms 6-(4-hydroxy-3-methylbutylamino)purine, whose trivial name is dihydrozeatin (DZ) (3Mok D.W. Mok M.C. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001; 52: 89-118Crossref PubMed Scopus (894) Google Scholar) (see Fig. 1). Among the CK species, tZ is most commonly found in higher plants and has a high activity in bioassays (4Spiess L.D. Plant Physiol. 1975; 55: 583-585Crossref PubMed Google Scholar, 5Schmitz R.Y. Skoog F. Plant Physiol. 1972; 50: 702-705Crossref PubMed Google Scholar). In higher plants, the first step of CK biosynthesis is catalyzed by adenosine phosphate-isopentenyltransferase (IPT), which uses dimethylallyl diphosphate as an isoprene donor to prenylate adenosine phosphate. In Arabidopsis thaliana, seven IPT genes (AtIPT1 and AtIPT3–AtIPT8) have been identified (6Takei K. Sakakibara H. Sugiyama T. J. Biol. Chem. 2001; 276: 26405-26410Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar, 7Kakimoto T. Plant Cell Physiol. 2001; 42: 677-685Crossref PubMed Scopus (347) Google Scholar). Biochemical studies suggest that the AtIPTs preferentially utilize ADP or ATP rather than AMP, resulting in the production of iP riboside 5′-diphosphate (iPRDP) or iP riboside 5′-triphosphate (iPRTP), respectively (7Kakimoto T. Plant Cell Physiol. 2001; 42: 677-685Crossref PubMed Scopus (347) Google Scholar, 8Takei K. Yamaya T. Sakakibara H. J. Plant Res. 2003; 116: 265-269Crossref PubMed Scopus (23) Google Scholar) (see Fig. 1). The low affinity of the AtIPTs for AMP implies that most of the natural iP riboside 5′-monophosphate (iPRMP) is formed by dephosphorylation of iPRDP and iPRTP, phosphorylation of iP riboside (iPR) by adenosine kinase, and conjugation of a phosphoribosyl moiety to iP by adenine phosphoribosyltransferase (3Mok D.W. Mok M.C. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001; 52: 89-118Crossref PubMed Scopus (894) Google Scholar) (see Fig. 1). The nucleotides are further dephosphorylated and deribosylated resulting in iPR and iP, respectively. A series of modifications of the adenine moiety of CKs occurs by reactions that are part of a purine metabolic pathway (3Mok D.W. Mok M.C. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001; 52: 89-118Crossref PubMed Scopus (894) Google Scholar). Classical bioassays (4Spiess L.D. Plant Physiol. 1975; 55: 583-585Crossref PubMed Google Scholar, 5Schmitz R.Y. Skoog F. Plant Physiol. 1972; 50: 702-705Crossref PubMed Google Scholar) and recent analyses of CK receptors in Arabidopsis (9Yamada H. Suzuki T. Terada K. Takei K. Ishikawa K. Miwa K. Yamashino T. Mizuno T. Plant Cell Physiol. 2001; 42: 1017-1023Crossref PubMed Scopus (393) Google Scholar, 10Inoue T. Higuchi M. Hashimoto Y. Seki M. Kobayashi M. Kato T. Tabata S. Shinozaki K. Kakimoto T. Nature. 2001; 409: 1060-1063Crossref PubMed Scopus (706) Google Scholar) and maize (11Yonekura-Sakakibara K. Kojima M. Yamaya T. Sakakibara H. Plant Physiol. 2004; 134: 1654-1661Crossref PubMed Scopus (161) Google Scholar) demonstrated that free base CKs, such as tZ and iP, are physiologically active forms. Moreover, differential ligand preferences of the receptors (11Yonekura-Sakakibara K. Kojima M. Yamaya T. Sakakibara H. Plant Physiol. 2004; 134: 1654-1661Crossref PubMed Scopus (161) Google Scholar) imply that side chain variations in isoprenoid CKs are highly significant for the diversification of the hormones' biological function. Biochemical and genetic mechanisms that control side chain variation have not been characterized well yet. At present, it is assumed that tZ could be synthesized via the iPRMP-dependent and the iPRMP-independent pathways (12Åstot C. Dolezal K. Nordström A. Wang Q. Kunkel T. Moritz T. Chua N.-H. Sandberg G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14778-14783Crossref PubMed Scopus (120) Google Scholar) (see Fig. 1). In the iPRMP-dependent pathway, tZ is formed by hydroxylation of iP-type CKs. The enzymatic activity hydroxylating the prenyl side chain was detected in a microsomal fraction of cauliflower (13Chen C.-M. Leisner S.M. Plant Physiol. 1984; 75: 442-446Crossref PubMed Google Scholar). It catalyzed the conversion of iP and iPR to tZ and tZ riboside (tZR), respectively; reactivities acting on the nucleotides were not examined (13Chen C.-M. Leisner S.M. Plant Physiol. 1984; 75: 442-446Crossref PubMed Google Scholar). Sensitivity to metyrapone, an inhibitor of cytochrome P450 monooxygenase (P450), and dependence of the activity on NADPH suggested that the reaction was catalyzed by a P450 (13Chen C.-M. Leisner S.M. Plant Physiol. 1984; 75: 442-446Crossref PubMed Google Scholar). However, this enzyme has not been further characterized. In the iPRMP-independent pathway, tZR 5′-phosphates are proposed to be produced directly by IPT using an unknown hydroxylated side chain precursor that may be derived from the cytosolic mevalonate (MVA) pathway (12Åstot C. Dolezal K. Nordström A. Wang Q. Kunkel T. Moritz T. Chua N.-H. Sandberg G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14778-14783Crossref PubMed Scopus (120) Google Scholar) (Fig. 1). The synthesis of tZR 5′-monophosphate (tZRMP) from AMP and 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate (HMBDP), a likely precursor, has been demonstrated to be catalyzed by TZS, an agrobacterial IPT (14Krall L. Raschke M. Zenk M.H. Baron C. FEBS Lett. 2002; 527: 315-318Crossref PubMed Scopus (52) Google Scholar). However, HMBDP was identified as a metabolic intermediate of the methylerythritol phosphate (MEP) pathway (15Hecht S. Eisenreich W. Adam P. Amslinger S. Kis K. Bacher A. Arigoni D. Rohdich F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14837-14842Crossref PubMed Scopus (198) Google Scholar), which occurs in bacteria and plastids (16Rohmer M. Nat. Prod. Rep. 1999; 16: 565-574Crossref PubMed Scopus (919) Google Scholar). Selective labeling experiments using 13C-labeled precursors specific for either the MEP or MVA pathway demonstrated that the isoprenoid side chain of iP and tZ predominantly originates from the MEP pathway, whereas a large fraction of the cZ side chain is derived from the MVA pathway (17Kasahara H. Takei K. Ueda N. Hishiyama S. Yamaya T. Kamiya Y. Yamaguchi S. Sakakibara H. J. Biol. Chem. 2004; 279: 14049-14054Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). However, the utilizing efficiency of Arabidopsis IPT was much lower for HMBDP than for dimethylallyl diphosphate in vitro (18Takei K. Dekishima Y. Eguchi T. Yamaya T. Sakakibara H. J. Plant Res. 2003; 116: 259-263Crossref PubMed Scopus (15) Google Scholar). Thus, the physiological significance of the iPRMP-independent pathway remains obscure to date. Although several routes might contribute to tZ production in higher plants, the physiological importance and functional differentiation of the possible pathways is unclear. As a first step toward an understanding of the trans-hydroxylation step of iP-type CKs in the iPRMP-dependent pathway, we attempted to identify CK hydroxylase genes in Arabidopsis. Here we demonstrate that CYP735A encode CK trans-hydroxylases that catalyze the biosynthesis of tZ and discuss the physiological role of the trans-hydroxylation in the regulation of CK metabolism and signaling. Construction of Plasmids—cDNA for an Arabidopsis NADPH-P450 reductase (ATR1) (19Urban P. Mignotte C. Kazmaier M. Delorme F. Pompon D. J. Biol. Chem. 1997; 272: 19176-19186Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar) was synthesized by reverse transcriptase-PCR and cloned into the NotI/SpeI site of the pESC-LEU vector (Stratagene) to yield a plasmid designated pESC-ATR1. The coding region of AtIPT4 was ligated into the ApaI/SalI site of pESC-ATR1 to yield a plasmid designated pESC-ATR1-IPT4. For preparation of the coding region of various P450s, we designed PCR primers based on predicted sequences from the Arabidopsis P450 data base (www.biobase.dk/P450/index.shtml). Each of the reverse transcriptase-PCR products was ligated into the pYES2.1/V5-His-TOPO vector (Invitrogen) to yield a series of plasmids designated pYES-P450. pYES2.1/V5-His/lacZ (Invitrogen) was used as a control plasmid (pYES-lacZ). Screening of CK Hydroxylase Genes—pESC-ATR1-IPT4 and each member of the pYES-P450 series were co-transformed into Saccharomyces cerevisiae strain YPH499 (Stratagene). The yeast cells were grown in complete minimal drop-out medium (20Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley-Interscience, New York1994: 13.1.1-13.1.7Google Scholar) without leucine and uracil (CM-Leu-Ura) and with 1% raffinose at 30 °C for 24 h. The liquid medium was changed to CM-Leu-Ura with 2% galactose, 1% raffinose, and 80 μg/ml 5-aminolevulinic acid, and the A600 was adjusted to 0.4 and further cultured overnight at 20 °C. After removal of cells by centrifugation, the supernatant was mixed with an equal volume of 2% acetic acid. The CK content was analyzed by liquid chromatographymass spectrometry (LC-MS). CK Analysis—CKs were analyzed with a LC-MS system (model 2695/ZQ2000MS; Waters) on a reverse-phase column (Symmetry C18, 5 μm, 2.1 mm × 150 mm; Waters) at a flow rate of 0.25 ml/min in 0.1% acetic acid with a linear gradient of methanol (0% for 1 min, 0–50% for 15 min, and 50–70% for 6 min). CKs were quantified in the selected ion recording mode. Monitored ions for each CK species were as follows: m/z 220 and 352 for tZR and cZ riboside, m/z 136 and 220 for tZ and cZ, m/z 222 and 354 for DZ riboside (DZR), m/z 136 and 222 for DZ, m/z 204 and 336 for iPR, m/z 136 and 204 for iP, and m/z 357 for [2H5]tZR. Other conditions for MS analysis were as described previously (8Takei K. Yamaya T. Sakakibara H. J. Plant Res. 2003; 116: 265-269Crossref PubMed Scopus (23) Google Scholar). Preparation of Microsomal Fraction—Yeast cells harboring pESC-ATR1 and pYES-CYP735As were cultured in 500 ml of medium under inductive conditions as described above. The microsomal fraction was prepared by the method of Venkateswarlu et al. (21Venkateswarlu K. Lamb D.C. Kelly D.E. Manning N.J. Kelly S.L. J. Biol. Chem. 1998; 273: 4492-4496Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), except that the buffer A did not contain reduced glutathione. After ultracentrifugation, the pellet was further washed once with the buffer A. The final preparation of microsomal pellet was resuspended in the buffer A. Protein concentration was determined with protein assay (Bio-Rad) using bovine serum albumin as the standard. The P450 content was estimated by the method of Omura and Sato (22Omura T. Sato R. J. Biol. Chem. 1964; 239: 2370-2378Abstract Full Text PDF PubMed Google Scholar). Synthesis of iP Nucleotides—iPRMP, iPRDP, and iPRTP were synthesized enzymatically with IPTs (AtIPT1 and TZS) from dimethylallyl diphosphate and with AMP, ADP, and ATP, respectively, and purified by anion exchange chromatography using a MonoQ column as described previously (8Takei K. Yamaya T. Sakakibara H. J. Plant Res. 2003; 116: 265-269Crossref PubMed Scopus (23) Google Scholar). tZRMP was purchased from Apex Organics (Devon, UK). Enzyme Assay—Microsomal proteins were incubated in 40 μl of reaction mixture (100 mm sodium phosphate, 10% sucrose, iP-type CK, 3 mm NADPH, 1 mg/ml bovine serum albumin, pH 7.5) at 20 °C for appropriate periods; the amounts of microsomal protein and molecular species of iP-type CK are indicated in the text. The reaction was terminated by the addition of 400 μl of termination buffer (50 mm CHES-NaOH, 0.5 mm MgCl2, pH 10.0). The mixture was treated with calf intestine alkaline phosphatase (10 units; Wako Pure Chemical, Osaka, Japan) at 37 °C for 30 min. After the addition of 20 μl of 20% acetic acid and 2 pmol [2H5]tZR (OlChemim, Olomouc, Czech Republic) to determine the recovery in the following steps, the mixture was centrifuged at 20,000 × g for 20 min. The supernatant was passed through a reverse-phase column (Oasis HLB, 10 mg/1cc; Waters) pre-equilibrated with 1 ml of methanol and 1 ml of water. The column was washed twice with 1 ml of water, and CKs were eluted with 1 ml of methanol. The solvent was evaporated under vacuum, and residual materials were dissolved in 0.1% acetic acid. After centrifugation, CKs in the supernatant were quantified with LC-MS. Identification of the Reaction Products as the Nucleotide Forms— Microsomal proteins (100 μg) were incubated in 200 μl of the reaction mixture with 50 μm iPRMP at 20 °C for 90 min. The reaction was stopped by adding 20 μl of 20% acetic acid. After centrifugation, the supernatant was passed through an Ultrafree MC centrifugal filter (Biomax-10; Millipore). The filtrate was loaded onto an ODS column (Supersphere RP-select B, 4 mm × 250 mm; Merck) by an HPLC system (model 600/717plus/PDA996; Waters) as described previously (23Takei K. Sakakibara H. Taniguchi M. Sugiyama T. Plant Cell Physiol. 2001; 42: 85-93Crossref PubMed Scopus (323) Google Scholar). The fraction containing the reaction product was dried and redissolved in 0.1% acetic acid. After centrifugation, the supernatant was analyzed with a LC-MS system as described above. The mass spectra were obtained by electrospray ionization scanning positive ions from m/z 120 to 500. The capillary voltage was 4 kV. Extraction of RNA—A. thaliana ecotype Columbia was grown hydroponically on MGRL culture medium (24Fujiwara T. Yokota-Hirai M. Chino M. Komeda Y. Naito S. Plant Physiol. 1992; 99: 263-268Crossref PubMed Scopus (293) Google Scholar) for 4 weeks as described (25Takei K. Ueda N. Aoki K. Kuromori T. Hirayama T. Shinozaki K. Yamaya T. Sakakibara H. Plant Cell Physiol. 2004; 45 (in press)Crossref PubMed Scopus (298) Google Scholar), and the rosette leaves, flowers, roots, and stems were harvested separately. The organs were soaked in water or 5 μm iP by vacuum infiltration and incubated for 1 h. For phytohormone treatment, Arabidopsis seedlings that had been grown on MGRL vertical agar plates for 12 days were sprayed with 5 μm of various hormones. After 1 h, total RNA was extracted with an RNeasy plant mini kit (Qiagen). Quantitative Real Time PCR—The real time PCR was performed on an ABIPRISM 7000 sequence detection system (Applied Biosystems) with a qPCR core kit for SYBR-Green I (Eurogentec) as described (25Takei K. Ueda N. Aoki K. Kuromori T. Hirayama T. Shinozaki K. Yamaya T. Sakakibara H. Plant Cell Physiol. 2004; 45 (in press)Crossref PubMed Scopus (298) Google Scholar). Sequences of the primers used were: CYP735A1, 5′-GGCCATGGTTTCGCAATC-3′ and 5′-CCGTTTCCGTTAAGCAAAGC-3′; CYP735A2, 5′-GCTCTTCCATCCACCACAACA-3′ and 5′-CGGATTGTGCTTCGTTAGCA-3′; ubiquitin10 (UBQ10), 5′-AACTTTGGTGGTTTGTGTTTTGG-3′ and 5′-TCGACTTGTCATTAGAAAGAAAGAGATAA-3′; Arabidopsis response regulator 5 (ARR5), 5′-TTTAAAAGCTCAAAGATTCACACACA-3′ and 5′-ATCAGCAAAAGAAGCCGTAATGT-3′. Isolation of Arabidopsis P450 Genes Involved in tZ Biosynthesis Using the AtIPT4/P450 Co-expression System—To identify gene(s) that catalyze tZ biosynthesis, we designed a screening method using an AtIPT4/P450 co-expression system in yeast. When AtIPT4, which catalyzes an initial step of the biosynthesis of iP nucleotides, was expressed in yeast, iP and iPR but not tZ and tZR were excreted into the culture medium (Table I, pYES-lacZ/pESC-ATR1-IPT4). Thus, AtIPT4 can synthesize iP nucleotides in yeast cells, whereas authentic yeast enzymes transform iP nucleotides into the corresponding nucleosides and free bases. However, the hydroxylation activity required for tZ production is lacking. We reasoned that if the appropriate P450 were co-expressed with AtIPT4 in yeast, tZ-type CKs could be expected to be synthesized. To construct such cell lines, we used two expression vectors, one carrying AtIPT4 and the other carrying one of a series of Arabidopsis P450s. To gain electrotransfer efficiency from NADPH to the plant P450s in yeast, an Arabidopsis NADPH-P450 reductase ATR1 was also expressed in the yeast cells in all cases (19Urban P. Mignotte C. Kazmaier M. Delorme F. Pompon D. J. Biol. Chem. 1997; 272: 19176-19186Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar).Table ICK levels in the culture medium of yeast expressing AtIPT4, ATR1, and CYP735AsPlasmidsCK levelsiPiPRtZtZRcZcZRDZDZRnmol/ml of culture mediumpYES-lacZ/pESC-ATR1-IPT4aControl plasmid.8.263.80NDbND, not detected at a detection limit of 0.01 nmol/ml.NDNDNDNDNDpYES-CYP735A1/pESC-ATR1-IPT4cExpression vectors for CYP735A1 or CYP735A2, respectively.3.711.753.462.070.020.011.891.71pYES-CYP735A2/pESC-ATR1-IPT4cExpression vectors for CYP735A1 or CYP735A2, respectively.5.912.912.851.320.060.020.710.51a Control plasmid.b ND, not detected at a detection limit of 0.01 nmol/ml.c Expression vectors for CYP735A1 or CYP735A2, respectively. Open table in a new tab The Arabidopsis genome contains more than 270 genes for P450s (Refs. 26Paquette S.M. Bak S. Feyereisen R. DNA Cell Biol. 2000; 19: 307-317Crossref PubMed Scopus (160) Google Scholar, 27Werck-Reichhart D. Bak S. Paquette S. Somerville C.R. Meyerowitz E.M. The Arabidopsis Book. American Society of Plant Biologists, Rockville, MD2002Google Scholar, 28Nelson D.R. Schuler M.A. Paquette S.M. Werck-Reichhart D. Bak S. Plant Physiol. 2004; 135: 756-772Crossref PubMed Scopus (357) Google Scholar; arabidopsis-p450.biotec.uiuc.edu; biobase.dk/P450/p450.shtml). For further experiments, we chose genes that appeared closely related to CK metabolism, either because they encoded homologues of P450s known to catalyze the trans-hydroxylation of the terminal-methyl group in isoprenoid compounds (At2g45560, At2g45580, At2g45550, and At3g52970) (29Collu G. Unver N. Peltenburg-Looman A.M. van der Heijden R. Verpoorte R. Memelink J. FEBS Lett. 2001; 508: 215-220Crossref PubMed Scopus (253) Google Scholar), because they were regulated by CKs (At1g67110) (30Rashotte A.M. Carson S.D. To J.P. Kieber J.J. Plant Physiol. 2003; 132: 1998-2011Crossref PubMed Scopus (237) Google Scholar) or because they were induced in a CK-overproducing mutant (At1g13710) (31Helliwell C.A. Chin-Atkins A.N. Wilson I.W. Chapple R. Dennis E.S. Chaudhury A. Plant Cell. 2001; 13: 2115-2125Crossref PubMed Scopus (151) Google Scholar). Their homologues (At5g38450, At2g46960, At2g46950, At4g27710, At5g09970, At1g01190, At3g61880, and At1g74110) and some others (At1g16410, At1g16400, At3g26220, At5g36220, At4g37310, At4g00360, At2g27690, At2g23180, and At2g34500) were also chosen. We expressed these P450s in the yeast system and found that the supernatants of cultures expressing CYP735A1 (At5g38450) and CYP735A2 (At1g67110) contained tZ and tZR at similar levels as iP and iPR (Table I). Significant amounts of DZ and DZR and traces of cZ and cZ riboside were also detected in both culture media (Table I). CYP735A1 and CYP735A2 (formerly named CYP709A1 and CYP709A2, respectively) are members of the CYP735A subfamily in Arabidopsis and share 79% identity at the amino acid level. CYP735A2 was first described as a CK-inducible gene by a DNA microarray analysis (30Rashotte A.M. Carson S.D. To J.P. Kieber J.J. Plant Physiol. 2003; 132: 1998-2011Crossref PubMed Scopus (237) Google Scholar). In a phylogenetic tree of Arabidopsis P450 genes, the CYP709B subfamily was a sister group of the CYP735A subfamily (biobase.dk/P450/p450.shtml). It consisted of three genes, CYP709B1 (At2g46960), CYP709B2 (At2g46950), and CYP709B3 (At4g27710), which showed 35–40% identity with the CYP735As at the amino acid level. We tested the ability of the CYP709Bs to hydroxylate CKs in the yeast system, but no secretion of tZ-type CKs was detected (data not shown). CYP735A1 and CYP735A2 Utilize iP Nucleotides as Substrates—Microsomal fractions prepared from yeast co-expressing ATR1 and CYP735A1 or CYP735A2 were used to measure the enzymatic activity in vitro. When iP nucleotides were used as substrates, efficient conversion to tZ-type CKs was detected (Fig. 2A). iPRMP and iPRDP were processed more efficiently than iPRTP by both CYP735As. The conversion efficiency of iP and iPR was less than 10% of that of iPRTP (Fig. 2A). These results strongly suggest that the CYP735As function as iP nucleotide hydroxylases in vivo. Although the activity was almost abolished when NADPH was absent from the reaction mixture, the presence of NADH partially restore the activity (Table II). This suggests that the reaction was not completely dependent on NADPH-P450 reductase, and NADH could act as an electron donor probably via other reduction systems such as a NADH-cytochrome b5 reductase/cytochrome b5 system (32de Vetten N. ter Horst J. van Schaik H.P. de Boer A. Mol J. Koes R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 778-783Crossref PubMed Scopus (139) Google Scholar). The substantial inhibition of the reaction by metyrapone was coincided with the sensitivity to this compound of the CK hydroxylase detected in the microsomal fraction of cauliflower (13Chen C.-M. Leisner S.M. Plant Physiol. 1984; 75: 442-446Crossref PubMed Google Scholar). CYP735A1 was more sensitive to metyrapone than CYP735A2 (Table II).Table IIEnzymatic properties of CYP735A1 and CYP735A2ConditionsRelative activityCYP735A1aMicrosomal fractions were prepared from yeasts harboring pESC-ATR1/pYES-CYP735As.CYP735A2aMicrosomal fractions were prepared from yeasts harboring pESC-ATR1/pYES-CYP735As.ControlbActivity was measured in the reaction mixture with 10 μg of microsomal protein and 10 μm iPRMP. The activities of CYP735A1 and CYP735A2 in control were 21.4 ± 0.5 and 0.57 ± 0.03 pmol/min, respectively (n = 3).100cRelative activity with the value in control taken as 100.100-NADPHdActivity was measured without NADPH.<0.11.0-NADPH + NADHeActivity was measured with 3 mm NADH.27.154.7+Me2SOf1% Me2SO was added to the reaction mixture.94.2108+1 mm metyraponeg1% Me2SO and metyrapone were added to the reaction mixture.12.050.5+10 mm metyraponeg1% Me2SO and metyrapone were added to the reaction mixture.1.26.7a Microsomal fractions were prepared from yeasts harboring pESC-ATR1/pYES-CYP735As.b Activity was measured in the reaction mixture with 10 μg of microsomal protein and 10 μm iPRMP. The activities of CYP735A1 and CYP735A2 in control were 21.4 ± 0.5 and 0.57 ± 0.03 pmol/min, respectively (n = 3).c Relative activity with the value in control taken as 100.d Activity was measured without NADPH.e Activity was measured with 3 mm NADH.f 1% Me2SO was added to the reaction mixture.g 1% Me2SO and metyrapone were added to the reaction mixture. Open table in a new tab In our routine method of measuring P450 activity, we dephosphorylated the primary products with phosphatase and monitored the nucleosides because of the difficulties to separate nucleotides on reverse-phase columns and the low sensitivity for nucleotides of our MS analysis. To confirm the nature of the primary product, the reaction products generated by CYP735A1 with iPRMP as a substrate were prepared on a large scale and were purified. The retention time determined by HPLC (Fig. 2B) and the spectrum obtained by LC-MS (Fig. 2C) showed that the primary product was tZRMP. We obtained a similar result with CYP735A2 (data not shown). CYP735A1 and CYP735A2 Catalyze trans-Hydroxylation in Vitro—In the culture medium of yeasts co-expressing AtIPT4/CYP735A, cZ-type and DZ-type CKs were also detected (Table I). To establish whether CYP735As catalyze the formation of cis-isomers and/or the reduction of the tZ side chain in vitro, we analyzed the reaction products by LC-MS. When iPRMP was used as substrate, both CYP735As mainly produced the trans-isomer; production of the cis-isomer occurred at a negligible level (Fig. 3, m/z 352). Although a small peak was detected at m/z 354, it was an unrelated peak because the retention time of the peak (13.3 min) did not coincide with that of the DZR standard (13.1 min; Fig. 3, m/z 354). When tZRMP was incubated with total soluble proteins prepared from the yeast transformants, production of DZR 5′-monophosphate was detected. However, this conversion was not CYP735A-dependent (data not shown). We concluded that the production of cZ and DZ-type CKs in the yeast expression system was due to endogenous enzymatic activities of the yeast cells and that CYP735As catalyze the stereo-specific formation of tZ nucleotides. Kinetic Parameters of CYP735A1 and CYP735A2—The contents of P450s in the microsomes were estimated to be 0.062 nmol/mg of microsomal protein for CYP735A1 by analysis of CO difference spectra (Fig. 4). The content of CYP735A2 could not be determined because of its low expression level in yeast (data not shown). We determined the kinetic parameters of CYP735A1 and CYP735A2 for the iP nucleotides (Table III). Of all substrates tested, the Km values of CYP735A1 and CYP735A2 for iPRMP were the smallest, whereas those for iPRTP were the largest. CYP735A2 exhibited higher substrate affinities than CYP735A1 in all cases. The Km values for iP and iPR could not be determined because of the low activities with these substrates. Furthermore, comparisons of the catalytic constants (kcat) and the specificity constants (kcat/Km) of the reactions strongly suggested that CYP735As preferentially act on iPRMP and iPRDP (Table III).Table IIIKinetic para
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