A Homolog of Old Yellow Enzyme in Tomato (1999)

A cDNA was isolated and characterized from a tomato shoot cDNA library, the deduced amino acid sequence of which exhibited similarity with yeast Old Yellow Enzymes (OYEs) and related enzymes of bacterial and plant origin. Sequence identity was particularly high with 12-oxophytodienoate 10,11-reductase (OPR) fromArabidopsis thaliana. The cDNA-encoded protein was expressed as a glutathione S-transferase fusion protein inEscherichia coli and was purified from bacterial extracts. The protein was found to be a flavoprotein catalyzing the NADPH-dependent reduction of the olefinic bond of α,β-unsaturated carbonyl compounds, including 12-oxophytodienoic acid. Thus, the tomato enzyme was termed LeOPR. The catalytic efficiency of LeOPR was highest withN-ethylmaleimide followed by 12-oxophytodienoic acid and maleic acid as substrates. Photoreduction of theLeOPR-bound FMN resulted in the formation of a red, anionic semiquinone prior to the formation of the fully reduced flavin dihydroquinone. Spectroscopic characterization of LeOPR revealed the formation of charge transfer complexes upon titration withpara-substituted phenolic compounds, a distinctive feature of the enzymes of the OYE family. The ligand binding properties were compared between LeOPR and OYE, and the findings are discussed with respect to structural differences between the active sites of OYE and LeOPR. A cDNA was isolated and characterized from a tomato shoot cDNA library, the deduced amino acid sequence of which exhibited similarity with yeast Old Yellow Enzymes (OYEs) and related enzymes of bacterial and plant origin. Sequence identity was particularly high with 12-oxophytodienoate 10,11-reductase (OPR) fromArabidopsis thaliana. The cDNA-encoded protein was expressed as a glutathione S-transferase fusion protein inEscherichia coli and was purified from bacterial extracts. The protein was found to be a flavoprotein catalyzing the NADPH-dependent reduction of the olefinic bond of α,β-unsaturated carbonyl compounds, including 12-oxophytodienoic acid. Thus, the tomato enzyme was termed LeOPR. The catalytic efficiency of LeOPR was highest withN-ethylmaleimide followed by 12-oxophytodienoic acid and maleic acid as substrates. Photoreduction of theLeOPR-bound FMN resulted in the formation of a red, anionic semiquinone prior to the formation of the fully reduced flavin dihydroquinone. Spectroscopic characterization of LeOPR revealed the formation of charge transfer complexes upon titration withpara-substituted phenolic compounds, a distinctive feature of the enzymes of the OYE family. The ligand binding properties were compared between LeOPR and OYE, and the findings are discussed with respect to structural differences between the active sites of OYE and LeOPR. Old Yellow Enzyme charge transfer glutathione S-transferase 12-oxophytodienoic acid 12-oxophytodienoate 10,11-reductase Old Yellow Enzyme (OYE)1(EC 1.6.99.1) was initially isolated from brewer's bottom yeast and was the first enzyme shown to possess a flavin cofactor (1Warburg O. Christian W. Naturwissenschaften. 1932; 20: 688Crossref Scopus (66) Google Scholar, 2Warburg O. Christian W. Biochem. Z. 1933; 266: 377-411Google Scholar). Despite extensive biochemical and spectroscopic characterization, the physiological role of the enzyme has remained obscure. OYE has been described as a diaphorase catalyzing the oxidation of NADPH in presence of molecular oxygen. However, molecular oxygen is commonly viewed as an opportunist electron acceptor, and the physiological oxidant has not been identified (3Massey V. Schopfer L.M. J. Biol. Chem. 1986; 261: 1215-1222Abstract Full Text PDF PubMed Google Scholar). A number of electron acceptors for reduced OYE are known including the olefinic bond of α,β-unsaturated ketones and aldehydes, suggesting that the physiological substrate may comprise such a structural feature (4Stott K. Saito K. Thiele D.J. Massey V. J. Biol. Chem. 1993; 268: 6097-6106Abstract Full Text PDF PubMed Google Scholar, 5Vaz A.D.N. Chakraborty S. Massey V. Biochemistry. 1995; 34: 4246-4256Crossref PubMed Scopus (133) Google Scholar). A growing family of OYE homologs has been identified in organisms with both prokaryotic and eukaryotic origins (4Stott K. Saito K. Thiele D.J. Massey V. J. Biol. Chem. 1993; 268: 6097-6106Abstract Full Text PDF PubMed Google Scholar, 6French C.E. Bruce N.C. Biochem. J. 1994; 301: 97-103Crossref PubMed Scopus (88) Google Scholar, 7Franklund C.V. Baron S.F. Hylemon P.B. J. Bacteriol. 1993; 175: 3002-3012Crossref PubMed Google Scholar, 8Miura K. Tomioka Y. Suzuki H. Yonezawa M. Hishinuma T. Mizugaki M. Biol. Pharm. Bull. 1997; 20: 110-112Crossref PubMed Scopus (76) Google Scholar, 9Buckman J. Miller S.M. Biochemistry. 1998; 37: 14326-14336Crossref PubMed Scopus (40) Google Scholar). Recently, the cloning and sequencing of a higher plant OYE homolog from Arabidopsis thaliana has been described (10Schaller F. Weiler E.W. J. Biol. Chem. 1997; 272: 28066-28072Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). This enzyme catalyzes the NADPH-dependent reduction of 12-oxophytodienoate (OPDA) to yield 3-oxo-2(2′(Z)-pentenyl)-cyclopentane-1-octanoic acid and was therefore termed 12-oxophytodienoate 10,11-reductase (OPR). The OPR-catalyzed reaction is part of the biosynthetic pathway to jasmonic acid, a compound with multiple hormonal activities in higher plants (11Weiler E.W. Laudert D. Schaller F. Stelmach B. Henning P. Romeo Phytochemical Signals and Plant-Microbe Interactions. Plenum Press, New York1998Google Scholar). Two isoforms of this enzyme (OPRI and OPRII) exist inArabidopsis and in Corydalis sempervirens (Rock Harlequin), only one of which (OPRII) reduces the 9S,13S enantiomer of OPDA, the precursor of biologically active 7-(epi) jasmonic acid (12Schaller F. Weiler E.W. Eur. J. Biochem. 1997; 245: 294-299Crossref PubMed Scopus (55) Google Scholar, 13Schaller F. Henning P. Weiler E.W. Plant Physiol. 1998; 118: 1345-1351Crossref PubMed Scopus (78) Google Scholar, 14Biesgen C. Weiler E.W. Planta. 1999; 208: 155-165Crossref PubMed Scopus (100) Google Scholar). In contrast, OPRI does not accept the isomers of OPDA with the 13S-configuration as substrates, and the physiological role of this isoform remains obscure (13Schaller F. Henning P. Weiler E.W. Plant Physiol. 1998; 118: 1345-1351Crossref PubMed Scopus (78) Google Scholar). We describe here the molecular cloning and overexpression in E. coli of an OPR from tomato plants, the purification of the recombinant enzyme, and the characterization of its substrate specificity. One of the most salient features of yeast OYE is its ability to form charge transfer (CT) complexes with a variety of aromatic and heteroaromatic compounds carrying an ionizable hydroxyl group. Formation of the CT complex results in a long wavelength optical transition in the absorbance spectrum. A positive correlation was observed between the absorbance maximum of the newly formed long wavelength transition and the Hammett para-constant of thep-substituted phenolic compounds, which can be considered as a measure of the phenolate ionization potential (15Massey V. Schopfer L.M. Dunham W.R. Bray R.C. Engel P.C. Mayhew S.G. Flavins and Flavoproteins. Walter de Gruyter & Co., Berlin1984: 191-210Crossref Google Scholar). This finding and the pH dependence of binding suggest that in the CT complex, the bound phenolate and the oxidized flavin act as the electron donor and acceptor, respectively. We report here on the spectral properties of the charge transfer complexes formed between recombinantLeOPR and phenolic ligands. This is the first detailed description of the properties of CT complexes formed by an OYE homolog isolated from a higher plant. All basic molecular techniques were adapted from Sambrook et al. (16Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) or Ausubelet al. (17Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley, New York1987: 247-269Google Scholar). A cDNA library was constructed from tomato (Lycopersicon esculentum cv. Castlemart II) shoot tissue in the yeast/E. coli shuttle vector pYES2 (Invitrogen, Groningen, The Netherlands). The LeOPR cDNA was isolated serendipitously by transformation of a yeast reporter strain 2J. Straßner and A. Schaller, unpublished data. using a selection system for proteases that interact with the peptide systemin (18Pearce G. Strydom D. Johnson S. Ryan C.A. Science. 1991; 253: 895-898Crossref PubMed Scopus (774) Google Scholar). The cDNA sequence of LeOPR was determined on both strands using fluorescent dideoxy chain terminators in the cycle sequencing reaction (Perkin-Elmer) and the Applied Biosystems model 373A DNA sequencer. Oligonucleotide sequencing and polymerase chain reaction primers were obtained from Microsynth (Balgach, Switzerland). The open reading frame of the LeOPR cDNA was amplified by polymerase chain reaction (Perkin-Elmer) (5′ primer, ATGGAAAATAAAGTCGTTGA; 3′-primer, GGGGTACCTCATGTCATGGTTTCTAGAA) and cloned into the StuI and KpnI restriction sites of pGEX-G (19Görlach J. Schmid J. Gene. 1996; 170: 145-146Crossref PubMed Scopus (12) Google Scholar), a derivative of pGEX-3X (Amersham Pharmacia Biotech). This vector allows expression of LeOPR in fusion with glutathione S-transferase under control of the isopropyl-1-thio-β-d-galactopyranoside-inducibletac promoter. The expression construct was verified by sequence analysis and was transformed into E. coli BL21. A 200-ml culture was grown from a single transformant and used to inoculate a 12-liter airlift fermentor (Bioengineering, Zürich, Switzerland). After 3 h at 30 °C, expression was induced by addition of isopropyl-1-thio-β-d-galactopyranoside to a final concentration of 1 mm. Cells were allowed to grow for another 6 h at 30 °C, when they were harvested by centrifugation and subsequently stored at −80 °C until purification of LeOPR. E. coli cells (10 g) were resuspended in 20 ml of Buffer A (50 mmTris-HCl, pH 8.0, 10 mm NaCl, 1 mm EDTA) containing 0.1 mg/ml DNaseI, 1 mg/ml lysozym, and 1 mmphenylmethylsulfonyl fluoride. After 20 min at room temperature, cells were lysed by sonication. The cell debris was removed by centrifugation (25000 × g for 10 min at 4 and the was to on a (Amersham Pharmacia in Buffer A. After extensive with Buffer A and Buffer (50 mm pH the fusion protein was with Buffer containing mm reduced and was used at a of with respect to h at room in to the fusion protein at the LeOPR with the The of was by LeOPR was purified by on as to the The final purification using a (Amersham Pharmacia a of in 20 mm pH 8.0, and system (Amersham Pharmacia Biotech). The of protein purification was by on using the system described by PubMed Scopus Google Scholar). were for using was isolated from of tomato plants using a (16Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). was on and to to LeOPR were on the using the cDNA DNA as a The and were as described A. C. Plant 1999; Google Scholar). were using a and the DNA was from tomato tissue using the DNA (Amersham Pharmacia Biotech). of DNA were using the enzymes in the to 3 and were to DNA analysis to (16Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The DNA were with the cDNA and at to be 20 and °C the of the DNA respectively. The were as described for were obtained by the oxidation of NADPH at in a Zürich, the substrate were at °C in 1 ml of mm pH including was at from to on the reaction observed with the The concentration of was determined at using a of The was determined by a of the maximum at of purified with that of FMN from the enzyme upon in presence of The reaction was upon the addition of the of the substrate were in to be from In the final concentration of in the was the of substrate were in presence of an system mm 10 of The was used for of the observed reaction were for and LeOPR in mm at pH were between and using a Photoreduction was under in was used as the electron the was using a A cDNA was isolated from a tomato shoot cDNA library, and both strands were number The cDNA an open reading frame of in that is by an that the cDNA the protein However, the the in is the at the does not the of the an its The amino acid sequence deduced from the open reading frame was found to be most related to that of OPR from A. thaliana (10Schaller F. Weiler E.W. J. Biol. Chem. 1997; 272: 28066-28072Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). as long as its physiological substrate remains to the protein Sequence of and were observed with enzymes of and bacterial respectively. A sequence of the amino acid sequence deduced from the tomato of and of is shown in sequence identity was observed the of the deduced protein Two of the amino acid in substrate binding are between the plant and yeast and to yeast as shown in the are by 1 in the OYE the is by a in LeOPR as as in most of the analysis 3 revealed the presence of LeOPR of tomato were highest in cells and in and were in The analysis of tomato DNA is shown in B. The observed restriction of the DNA with revealed and of The of the observed for DNA to from the cDNA to the of at one related to that of LeOPR in of amino acid OPR yeast and plant OYE The of number and and and OYE homologs from A. thaliana number and number are compared with that of LeOPR number The was with the amino acid that are in the of in a acid in binding of phenolic ligand and in the of the anionic flavin semiquinone are of the active of OYE and OPR A the amino the cofactor FMN and the bound phenolic ligand as by and 1994; Full Text Full Text PDF PubMed Scopus Google Scholar) on this a model of the active of LeOPR using the sequence of amino are shown in and amino acid are shown In to yeast OYE, the amino acid were found in LeOPR at and that amino acid in the yeast OYE has in the plant homolog in the shown in The was used to the structural LeOPR was cloned into the expression vector pGEX-G (19Görlach J. Schmid J. Gene. 1996; 170: 145-146Crossref PubMed Scopus (12) Google Scholar) and coli in fusion with 1 of E. coli of were purified by was from the by using and was purified by this of revealed the presence of of and The of the and its molecular is in with the of for LeOPR deduced from the cDNA The amino acid of the were determined and found to to the and to an sequence respectively. the a form of LeOPR and is most a OYE is known to be a protein in its L.M. Massey V. A of Press, 247-269Google Scholar). of LeOPR and its and is to be to the formation of between the LeOPR and the fusion protein were found to the oxidation of NADPH in presence of molecular oxygen. the for of OYE, the bond in α,β-unsaturated carbonyl compounds was found to be an electron (4Stott K. Saito K. Thiele D.J. Massey V. J. Biol. Chem. 1993; 268: 6097-6106Abstract Full Text PDF PubMed Google Scholar, 5Vaz A.D.N. Chakraborty S. Massey V. Biochemistry. 1995; 34: 4246-4256Crossref PubMed Scopus (133) Google Scholar, L.M. Massey V. A of Press, 247-269Google Scholar). observed an of NADPH oxidation by LeOPR in presence of α,β-unsaturated carbonyl the oxidation of NADPH is to the of the carbonyl and the molecular oxygen to the observed reaction to molecular oxygen as a potential electron acceptor, the of with α,β-unsaturated carbonyl was in presence of an system and were from for a selection of α,β-unsaturated carbonyl compounds and are in was found to accept a of α,β-unsaturated The highest catalytic efficiency was observed × The and its the found in which is the substrate of the OYE homolog in 12-oxophytodienoate acid, was reduced by LeOPR with a catalytic efficiency of × acid was found to be a its acid was not at The catalytic were for from to × A of and acid that the and the of the at the are for substrate properties of at a concentration of at a concentration of at a concentration of at a concentration of at a concentration of at a concentration of were from were at °C in 1 ml of mm pH including 20 mm and 10 of The was used for the of the The at a concentration of 30 in a were from were at °C in 1 ml of mm pH including 20 mm and 10 of The was used for the of the data. LeOPR and were with a of in to the of the protein to form CT complexes to for OYE Massey V. J. Biol. Chem. Full Text PDF PubMed Google Scholar). A titration is shown in addition of to a long wavelength absorbance with a maximum at as a of ligand spectral in the and of the flavin were The formation of charge transfer results in in the absorbance of the The spectral of the CT complexes formed between and as as are in the of the CT complexes were for LeOPR in to both long wavelength absorbance A was observed between the absorbance of the long wavelength transition and the the of the CT complex be to the phenolic a that is the phenolate that to the enzyme and that the complex, the phenolate and the flavin act as the charge donor and acceptor, of LeOPR and with as by and Massey para-constant as by and et al. absorbance of LeOPR and were upon titration with the The absorbance of the that as a of ligand concentration are as are the of the charge transfer complexes and the from the titration as by and Massey Massey V. J. Biol. Chem. Full Text PDF PubMed Google Hammett para-constant as by Chem. Scopus Google Scholar) and et al. C. A. Chem. 1991; Scopus Google Scholar). in a of the to the absorbance maximum of the charge transfer The of the absorbance of the long wavelength were the as by Chem. Scopus Google Scholar) and et al. C. A. Chem. 1991; Scopus Google of the The used for and OYE CT complexes Massey V. J. Biol. Chem. Full Text PDF PubMed Google Scholar) are by and open was with the and the are shown by CT and a CT The absorbance of LeOPR and were upon titration with the The absorbance of the that as a of ligand concentration are as are the of the charge transfer complexes and the from the titration of FMN was under by with in the presence of as a of the reduction of the flavin cofactor resulted in a in the absorbance at the absorbance maximum with an at of the formation of a red, anionic flavin that the flavin semiquinone is the only flavin at an of for the semiquinone at this The is to that observed for the flavin semiquinone R.C. Massey V. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of the flavin semiquinone was followed by a reduction to the fully reduced a of the absorbance at the of flavin semiquinone was to be formed In the presence of the electron flavin semiquinone was that the is Recently, plant high similarity with yeast OYE were characterized from C. sempervirens and cloned and they were found to the reduction of (10Schaller F. Weiler E.W. J. Biol. Chem. 1997; 272: 28066-28072Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, F. Weiler E.W. Eur. J. Biochem. 1997; 245: 294-299Crossref PubMed Scopus (55) Google Scholar). termed with which is an of the pathway in the of jasmonic acid. the the of an catalyzing the reduction of the OPDA (13Schaller F. Henning P. Weiler E.W. Plant Physiol. 1998; 118: 1345-1351Crossref PubMed Scopus (78) Google Scholar). was to be the in jasmonic acid the role of OPRI in the pathway is The may be by of the and a role for OPRI has been in a of OPDA for the of jasmonic acid (13Schaller F. Henning P. Weiler E.W. Plant Physiol. 1998; 118: 1345-1351Crossref PubMed Scopus (78) Google cloned and characterized was found to OPDA, which is the of the of Schaller and E. unpublished data. that the of LeOPR that of OPRI sempervirens and A. reduces the However, analysis revealed the presence of at related the tomato the The and characterization of the LeOPR to in C. sempervirens be in the of in plant the substrate of LeOPR is to that physiological compounds may be reduced by this a of with the basic substrate an α,β-unsaturated carbonyl and physiological of LeOPR may In of this remains to be the for physiological and OPRI with respect to substrate of yeast OYE, of A distinctive feature of yeast OYE is the formation of CT complexes with phenolic Massey V. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Massey V. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Massey V. J. Biol. Chem. Full Text PDF PubMed Google Scholar). We here that a of phenolic form CT complexes with LeOPR. This is the first of such complexes with a plant biochemical for the between yeast and plant homologs that was (10Schaller F. Weiler E.W. J. Biol. Chem. 1997; 272: 28066-28072Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). The absorbance and the of CT complexes were for OYE the were higher for the CT complexes with LeOPR. Despite a of the charge transfer absorbance the Hammett of the at the aromatic of the phenolic a dependence with a to the one obtained for OYE This that the flavin as the and the phenolic ligand as the donor in CT as was for yeast OYE Massey V. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of the CT complex absorbance in of LeOPR that the between the molecular of FMN and the highest molecular of the phenolic ligand is for LeOPR for yeast In the potential of the FMN is the potential of the phenolic ligand is positive in LeOPR as compared with yeast OYE CT to the of yeast is bound on the of the flavin system 1994; Full Text Full Text PDF PubMed Scopus Google Scholar) The a bond to the hydroxyl of the chain of the is in bond with the of and respectively. is in LeOPR and is in plant and yeast with the of the OYE homologs is the only the is by a in of the OYE protein a to the is by a in this A of the found is shown in in on the known of yeast OYE the amino acid and were into the that the and in LeOPR a of the bond in the binding of the phenolic which may be for the higher and the properties of the charge transfer between the flavin and the phenolic ligands. of with in was to in a yield of protein Z. Massey V. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). was that the with flavin in the yield of Z. Massey V. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). and plant the amino acid may for the The may in a higher in this part of the for a in the binding findings that the does not the formation of CT the formation of CT complexes can be for of the OYE family a an in and that the structural is to as discussed between the of OYE and LeOPR is the in LeOPR of a to in This is from the sequence shown in a to OYE to be in this to for an with the yeast The active to be to that of of LeOPR results in the formation of an anionic flavin semiquinone prior to the reduction to the flavin Formation of the flavin semiquinone was for yeast OYE R.C. Massey V. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of an anionic flavin semiquinone in the flavin binding is to the presence of the chain of an or in the of the of the flavin which the for the formation of a charge in this V. S. Moore J. Biol. Chem. Full Text PDF PubMed Google Scholar, S. Massey V. Eur. J. Biochem. PubMed Scopus Google Scholar). In the of yeast OYE the presence of an and of a in of the of the flavin amino are in yeast and the plant homologs and the of an anionic flavin semiquinone in can be by the presence of an flavin The of the anionic flavin semiquinone in as was shown in in the presence of an electron anionic semiquinone be In of OYE, of the anionic flavin semiquinone was in the presence of an electron that the of is R.C. Massey V. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of of the anionic flavin semiquinone is a of the one electron potential of and potential of In yeast OYE, are by 30 R.C. Massey V. J. Biol. Chem. Full Text PDF PubMed Google to the observed In of the of a flavin semiquinone in presence of an electron that the of the flavin semiquinone is as compared with The of is We for in the of and W. Weiler and Schaller for a analysis and