Molecular Cloning and Characterization of a cDNA Encoding Pea

VOl. 269,No.49, Issue of December 9, pp, 31129-31133, 1994 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Molecular Cloning and Characterization of a cDNA Encoding Pea Monodehydroascorbate Reductase* (Received for publication, July 1, 1994)

Siva Subramanian Murthy and BarbaraA. ZilinskasS From the Department of Plant Science, Cook College, Rutgers University, New Brunswick, New Jersey 08903-0231 generating monodehydroascorbate free radical (MDA).’ MonoMonodehydroascorbate radicals are generated in plant cellsenzymatically bythehydrogenperoxide dehydroascorbate can be directly reduced to ascorbate by the scavenging enzyme, ascorbate peroxidase, and nonenzyenzyme monodehydroascorbate reductase (MDAR; EC 1.6.5.4) matically via the univalent oxidation of ascorbate by in the reaction superoxide,hydroxyl,andvariousorganicradicals. monodehydroascorbate + NAD(P)H”-f ascorbate + NAD(P)’. Regenerationofascorbate is achieved bymonodehydroascorbate reductase (EC 1.6.5.4) using NAD(P)H Alternatively, MDA can disproportionate t o ascorbate and deas an electron donor or, alternatively, by a set of two hydroascorbate. The latter is then reduced back to ascorbate coupled reactions requiring dehydroascorbate reducusing reduced glutathione, which, in turn, is regenerated cattase, glutathione reductase, glutathione, and NAD(P)H. As monodehydroascorbate reductaseis a key enzymein alytically by glutathione reductase with electrons provided by maintaining reduced pools of ascorbate, an important NAD(P)H. The MDA free radical can also be generated enzymatically by antioxidant,weundertookthisstudytolearnmore ascorbate oxidase (3) and by a variety of nonenzymatic reacabout its structure, function, and regulation. Herein we report the molecular cloning and characterization of a tions including oxidation of ascorbate by superoxide and hyof droxyl radicals (4), reaction of ascorbate with products of the cDNA encodingmonodehydroascorbatereductase pea (Pisurnsatiuurn L.). The cDNA encodes a 433-amino reaction of lipid peroxyl radicals and a-tocopherol (51, and the its acid polypeptide that shows, respectively, 73 and 87% metal-catalyzed autoxidationof ascorbate (6). In addition to identity with peptide fragments from soybean and cukey involvement in reactive oxygen scavenging, MDAR has cumber monodehydroascorbate reductase. Monodehybeen implicated to play a central role in plant cell elongation droascorbate reductase contains the NAD(P)H and FAD (71, mediating the transport of electrons through the transbinding domains of other flavin oxidoreductases. The plasma membrane redox system (8). cloned enzyme lacksa transit peptide, but the sequence Monodehydroascorbate reductase has been shown to be of the carboxyl terminus is Ser-Lys-Ile, similar to the widely distributed phylogenetically; it has been found in nutargeting motif found in peroxisomal proteins. When ex- merous plants, animals, and protists(9). Within plants, it has pressedinEscherichia coli fusedtomaltose-binding been found in chloroplasts,glyoxysomes, mitochondria, and the protein,monodehydroascorbatereductasehasenzycytosol (101, which is consistent with the fact that MDA radical matic properties comparable with purified soybean and seems to be ubiquitous in thecell. The enzyme has been puricucumber monodehydroascorbate reductase. Northern blot analysis shows that the monodehydroascorbate re-fied to homogeneity from cucumberfruit (11)and soybean root is expressed nodules (12) and hasbeen shown t o be a FAD-containing monductase transcriptis 1.6 kilobase in size and omer with a molecular mass of 47 and 39 kDa, respectively. at relatively low levels in all plant tissues examined. These MDARs differ from the 66-kDa MDAR purified from Neurospora, which does not contain flavin (13). Despite the expected physiological significance of MDAR in mammals, the The ascorbate-glutathione pathway, also known as the Hal- enzyme has not been characterizedfully, although the activity liwell-Asada pathway for its codiscoverers (1,2), has long been has been detected in microsomes and mitochondria (14 and recognized to play a central role in scavenging reactive oxygen references cited therein). As little is known about the structure species in plants. Although this pathway was initially impli- of MDAR or the regulationof its expression, we sought toclone on the cated to be only functional in chloroplasts, it is now clear that a cDNA encoding this enzyme to enable detailed studies it exists in various subcellular compartments. In the first stepcharacterization of the enzyme and itsfunction and regulation of the pathway, hydrogen peroxide is reduced by the enzyme under normal conditions and in response to oxidative stress. ascorbate peroxidase, using ascorbate as electron donor and Herein we report themolecular cloning and characterizationof a cDNA encoding MDAR from pea. MATERIALS AND METHODS * This work was supported in part by state funds and the United States Hatch Act, by Grant US-2208-92 from the United States-Israel Generation of a MDAR-specificProbe by the Polymerase Chain ReacBinationalAgnculturalandDevelopmentalFund,and by Grant tion (PCR)-AMDAR-specificprobewas generated by the PCRon a R 820010-01-0 from the United States EnvironmentalProtection m m ) provided by Clontech. Agency. This is New Jersey Agricultural Experiment Station Publica- cDNA pool from soybean seedlings (Glycine Three specific degenerate primers were synthesized in the Millipore costs of publication of this article were defrayed tion D-01909-1-94. The Cyclone DNA synthesizer:one (primer 1in Table I) corresponding tothe in part by the payment of page charges. This article must thereforebe amino-terminalamino acid sequence of soybean (12) and the other two hereby marked “aduertisement” in accordance with 18 U.S.C. Section (primers 2 and 3, Table I) derived from two different peptidefragments 1734 solely to indicate this fact. The nucleotide seguence(s) reported in this paper has been submitted from the carboxyl-terminaldomain of cucumber(Cucumus satiuus) to the GenBankTM/EMBL Data Bank with accession number(s) U06461. $ To whom correspondence shouldbe addressed: Plant Science Dept., The abbreviations used are: MDA, monodehydroascorbate; MDAR, 207 Lipman Hall, Cook College, Rutgers University, New Brunswick, monodehydroascorbate reductase; PCR, polymerase chainreaction; bp, N J 08903-0231. Tel.: 908-932-8866; Fax: 908-932-8899. base pair(s); MBP, maltose-binding protein.

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Pea Monodehydroascorbate Reductase cDNA TABLEI Partial MDAR amino acid sequences from which PCR primers were designed Amino acid sequence

Primer 1. Primer 2. Primer 3. Primer 4. Primer 5.

MAKTFKYI ( 1 2 ) YAVGDVA ( 15 ) WQFYGDN ( 15 ) VHSFXYI SKI**

Primer sequence

5"CCGAATCATGGCHAHTTYAARTAYAT-3'

5'-AATCTAGAGCNACRTCNCCNACDGCRTA-3' 5"AATCTAGARTTRTCNCCRTARAAYTGCAA"'

5'-GGCGTACGTACATTCGTTCAAGTAT-3' 5'-CCCCTCTAGATCATTATTTTACT-3'

MDAR (15). To facilitate subsequent cloning, the amino-terminal and mM Tris-HC1, pH 7.8, containing 10 mM /3-mercaptoethanol and 50 mM carboxyl-terminal primers were designed respectively with EcoRI and EDTA. The cells were centrifuged again and resuspended50in ml of the XbaI restriction sites at their 5' ends. The PCR was performed under same buffer. The resuspended cells were sonicatedfor a total of 3 min the following conditions: the first cycle a t 95 "C for 5 min, 37 "C for 2 (30 s on, 30 s off) at 4 "C. The sonicated cells were then centrifuged at min, and 72 "C for 3 min; the next fourcycles at 95 "C for 1 min, 37 "C 17,000 x g for 30 min. The supernatant, designated as crude extract, for 2 min, and 72 "C for 3 min; the following 34 cycles at 95 "C for 1min, was affinity purified through an amylose column as described previ50 "C for 2 min, and 72 "C for 3 min; and the last cycle a t 95 "C for 1 ously (18). All steps were performeda t 4 "C. A similar preparationfrom min, 50 "C for 2 min, and 72 "C for 3 min. The PCR products were E. coli 745 transformed with the intact pMAL-c2 vector was used as a purified from an agarose gel using glassmilk (Geneclean, BIO 101, Inc.) control. Protein concentration was estimated by the method of Bradford and cloned into the pKS13 pBluescriptvector (Stratagene). (191, and denatured samples were analyzed by SDS-polyacrylamidegel Screening of the Agtll cDNA Library-A cDNA library, constructed electrophoresis (20) using a 10% polyacrylamide resolving gel. from pea leaf mRNA in the expression vector Agtll, was generously MDAR activity assays on the affinity-purified fusion protein were provided by Dr. J. Stephen Gantt (University of Minnesota). The library performed as described previously (11) with some modifications. The was used to infect the host strain Escherichia coli Y1090, and approxi- assay was carried out in a final volume of 1 ml containing 50 mM mately 7 x lo7 plaques were screened with the 700-bp PCR product Tris-HC1, pH 7.8, 1.0 mM ascorbate, 0.1mM NADH, 0.275 unit of ascorobtained using primers 1and 2 (see Table I). Plaques were transferred bate oxidase (Boehringer Mannheim) to generate 2.0 p~ MDA, and to nylon membranes (MAGNA,M.S.I), and the DNAwas denatured andsufficient fusion protein togive a change in A,,, of 0.02-0.15/min. The neutralized as described previously (16) and immobilized with a Strat- reaction was initiated by the addition of the fusion protein. Assaysfor agene W cross-linker. Filters were prehybridized at 50 "C for 4 h in 6 determining K, for NADH, NADPH, and MDA contained, respectively, x SSC, 0.1% SDS, 0.2 mg/ml bovine serum albumin, 0.2 mgiml Ficoll, 0-20 pmol of NADH, e 1 7 5 pmol of NADPH, and 0-3 pmol of MDA. The and 0.2mg/ml denatured calf thymus DNA. Following prehybridization, maximum concentration of MDA which could be generated using the the filters were incubated with the 3ZP-labeled 700-bp, gel-purified PCR ascorbate/ascorbate oxidase was 3 pmol. The apparent K,,, and V, product (specific activity of 1 x lo5cpm/ml of hybridization solution)for values were obtainedby fitting the initial reactionvelocity uersus sub16 h under the same conditions. Random primer labeling of the product strate concentration data directly to the Michaelis-Menten equation by was performed with[ ( Y - ~ ~ P I ~ A T P radiolabeled as the nucleotide usinga nonlinear regression analysis. random primer labeling kit (U. S. Biochemical Corp.). The filters were Northern Blot Analysis-RNA was isolatedfrom roots and the youngwashed three times (10 min each wash) with x SSC2 and0.2% SDS and est and oldest leaves of 10-day postemergence pea seedlings as desubsequently three times (10 min each wash) with1 x SSC and 0.2% scribed by Chomczynski and Sacchi (21) and quantified spectrophotometriSDS. The filters were exposed to X-Omat AR film (Kodak). Putative cally. In addition, flowers were collected from 30-day postemergence pea positive clones were rescreenedtwice at a lower density to obtain single plants. Plants were grown in the greenhouse and watered with halfpurified plaques. strength Hoagland's solution. RNA was also isolatedfrom etiolated pea Nucleotide Sequence Analysis-The nucleotidesequence of the leaf tissue from plants grownfor 10 days in complete darkness. Thirty MDAR cDNA was determined by dideoxy sequencing (17) of denatured pg of total RNA was subjected to electrophoresis on a 1.2% agarose gel plasmid templates using T7 DNA polymerase (U.S. Biochemical Corp.) containing 2.2 M formaldehyde, transferred to Duralon-W membranes and w3'S-dATP as the radiolabeled nucleotide. Plasmids were isolated (Stratagene), and prehybridized with the prehybridization solution deusing a Qiagen plasmidkit. Both strands were sequenced without am- scribed above at 65"C for 4 h. The filters were then hybridized with the biguities by progressive sequencing with synthetic 17-base oligonucleo- 32P-labeled, full-length MDARcDNA probe for 16 h at 65 "C. Subseat 60 "C with 0.1 x SSC and tide primers. Sequencing reactions were analyzedon a denaturing 6% quently, the filters were washed six times (w/v) polyacrylamide gel. Comparison of the deduced amino acid se- 0.2% SDS for 15 min each wash and then exposed to x-ray film. The peaS rRNAPCR-derived quence with sequences in the GenBank data base was done using the same blot was subsequently hybridized to a 18 GCG version 7.2 sequence analysis software. DNA probe (22) as an internal control t o assess RNA loading. Hybrida n Ultrascan XL laser densitometer Subcloning of the MDAR Coding Sequence into the Expression Vector ization signals were analyzed using pMAL-c2-The MDAR coding sequence was subcloned into the expres-(Pharmacia Biotech Inc.). sion vector pMAL-c2 (New England Biolabs) as follows. Two oligonuRESULTS AND DISCUSSION cleotides were synthesized correspondingthe to amino-terminal (primer 4) and carboxyl-terminal (primer 5) regions of the MDAR open reading Cloning and Sequence Analysis of Pea MDAR cDNA-The frame (refer toTable I). SnaBI and XbaI restriction sites were incorpo- availability of partial amino acid sequences obtained from purated, respectively, into the primerst o facilitate directional cloning into rified soybean (12) and cucumber (15) MDAR enabled us to the XmnI and XbaI sites of pMAL-c2. With the MDAR cDNA template in pBluescript, the PCR was carried out as follows: the first cycle at clone a full-length cDNA encoding pea MDAR. Sixteen plaques 95 "C for 5 min, 50 "C for 2 min, and 72"C for 3 min; 33 cycles at 95 "C hybridized per 10,000 plaques screened. Phage containing the for 1 min, 50 "C for 2 min, and 72 "C for 3 min; and the last cycle at largest cDNA insert were plaque purified and used to isolate a 95 "C for 1min, 50 "C for 2 min, and72 "C for 3 min. The 1,299-bp PCR 1,567-bp cDNA for further analysis. The cDNA was subcloned product was gel purified, subsequently digested withSnaBI andXbaZ, into pBluescript pKS13 (Stratagene) andsequenced using synand ethanol precipitated. ThepMAL-c2 vector was digested with XmnI thetic primerscorresponding to theplasmid vector and internal and XbaI, gel purified, ligated with the PCR product, and used t o transform a protease-deficient strain of E. coli (E. coli 745,New Eng- sites inMDAR cDNA (as they were determined). Both strands land Biolabs). The recombinants were characterized by restriction anal- were sequenced without ambiguity. The nucleotide sequence ysis and by expression of the maltose-binding protein (MBP).MDAR and deduced amino acid sequence of the cDNA are shown in fusion protein. Fig. 1. The cDNA contains a n open reading frame of 1,299 bp Analysis of the MBP.MDAR Fusion Protein-A 1-liter culture of re- encoding a protein of 47,000 M,, in agreement with the M , of combinant cells was grown at 37 "C to a n A,,,of -0.5 in LB broth 47,000 as determined by subsequent SDS-polyacrylamide gel containing 125pg/pl ampicillin. Isopropyl-1-thio-/3-D-galactopyranoside was then added toa final concentration of 0.3 mM, and the cells were electrophoresis of the expressed protein and comparable to the allowed to grow for an additional 2 h. The cells were then pelletedat M , of purified cucumberand soybean MDAR (11,121.A putative 3,000 x g for 15 min and washedonce with chilled extraction buffer, 50 polyadenylation signal (AATAAA) appears 30 bp upstream

Pea Monodehydroascorbate Reductase cDNA ggcaaaaagccgaagttgcagcgattttcatagcaatcaaaaatggtgcattcgttcaag 1

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tatatcatcattggaggaggagtttcagctggttatgcagcaagggagtttgtgaaacaa 61

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ggagttcatcctggggagcttgcaattatatctaaagaagcggtagcaccttatgaacgt

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cctgctctgaqcaaggcttacctttttccaqagtctcctgctaggcttcctyggttccat

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acctgtgttggaagtgggggagaaagattgcttccagagtggtacagtgagaaagggata

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cayctgtatctcagtacagaaattgtaagtgcagatcttgctgcaaaatttctgaaaagt

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gcaaacggagaacacttcgattaccagactttggttatcgcaacaggctcagctgttata

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aggttgacagatttcggtgtaataggagctaatgccaaaaacatattttaccttagygag """".+""""~+--------"--------+"""...+"""".+

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gttgatgatgctgacaaattgtacgaggcaatcaaaagaaagaagaatgcgaaacgtgtg 481

"""".+""""~+"--"--"-----"".+"."....+""."..+

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gttgttqgaggaggatacattggtctggagttgagtgcagttttgaaactcaatgatctt 541

"""".+""""~+""-"-"----""+.".."..+"""."+

Pea 251 ADIVIVGVGG RPQISLFKGQ VEEQHGGIKT DSFFKTSVPD VYAVGDVATF Cucumber ADIVVVGVGG RPLVSLFK TSIPD VYAVGDVATY 301 PLKLYNDVRR VEHVDHARKS AEQAAKAIFA ADVGKSVEEY DYLPYFYSRS Pea Cucumber PLKLYNELRR VEHVDHARLK SIEEY DYLPYFYSRT

Pea 351 FDLSWQFYGD NVGETVLFGD NDPASSKPKF GTYWIKEGKV VGAFLEGGTP Cucumber FNLAWQFYGD NVGETVLFGD N F GTYWIK Pea 4 0 1 DENKAIAKVA RAKPAVEDVN QLAEEGLSFA Cucumber VA DEY RVQPPVESLD OLAK

V VGVFLEGGTP

SKI

FIG. 2. Amino acid sequence comparison between pea MDAR and soybean and cucumber MDAR. Alignment of the deduced yatgttaccatggtctacccagaaccttggtgtatgccacgactttttacttctgaaatt """".+""""~+"----""----""+.."..".+""..."+ amino acid sequence of pea MDAR with homologous peptide fragments D V T M V Y P E P W C M P R L F T S E I from soybean (12) and cucumber MDAR (15). Y

601

EGYYANKGIN IIKGTVAVGF TANSDGEVKE VKLKDGRVLE LKDGRVLE

""""~+"""".+----"""+..".."~+""."..+""""~+

A 421

Pea 201 LFTSEIAAFY Cucumber

""""~+"""".+"-------t"+....."..+"""".+""."..+

Q 361

Pea 1 5 1 DKLYEAIKRK KNAKRVVVGG GYIGLELSAV LKLNDLDVTM VYPEPWCMPR Cucumber DQLVEAK

"""""""""""------"+""..".+""""""""""

T 301

Pea 1 0 1 AKFLKSANGE HFDYQTLVIA TGSAVIRLTD FGVIGANAKN IFYLREVDDA Cucumber N IFYLREIADA

""""~+"""".+"----""+"."....+..".""+"."....+

P 241

Pea 51 KAYLFPESPA RLPGFHTCVG SGGERLLPEW YSEKGIQLYL STEIVSADLA Cucumber K

"""".+"""".+".."...+""..."+."""..+"""".+

G 181

1 MVHSFKYI I 1 GGGVSAGYAAREFVKQGVHP GELAI ISKEA VAPYERPALS MAKTFKY II L G EA VAPYERPALS

""""~+"""".+"----""+""..".+"""".+"..."..+

Y 121

Pea Soybean Cucumber

""""~+"""".+""-"-"--"""..+".""..+""..."+

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gctgctttctatgagggatattatyccaataaaggyatcaatatcattaaagygacagtt 661

"""".+""""~+"....".+"."....+"...."~*"".....+

from the start of the poly(A) tail. The deduced amino acid sequence of pea MDAR shows, respectively, 87 and 73% idengctgttggattcactgccaactccgatggaqaggtaaaagaagtcaaactaaaggatggt 721 " " " " ~ + " " " " ~ + ~ ~ ~ " " " + " " - " ~ ~ . + " ~ ~ " ~ " + " " ~ ~ " . + tity with isolated peptide fragments from cucumber MDAR (15) A V G F T A N S D G E P K E V K L K D G and the amino-terminal region of soybean MDAR (12) deteragggtcctgqaagcagatattgtcattgttggtgttggaggaaggcctcaaatatcctta mined by Edman degradation (Fig. 2). These results indicate 781 " " " " ~ + " " " " ~ + ~ ~ ~ ~ . " " + ~ " " ~ ~ " + . " " " " + " ~ " ~ ~ " + R V L E A D I V I V G V G G R P Q I S L that the cloned cDNA does not encode a transit peptide, and therefore it is highly unlikely to encode a plastid or mitochonttcaaagggcaggttgaagagcagcatggtggaatcaagactgattccttcttcaaaaca 841 """"~+""""~+--------"-""."~+.""""+"""".+ drial isoform of MDAR. Rather, the cDNA probably encodes a F K G Q V E E Q H G G I K T D S F F K T cytosolic isoform of MDAR.We note, however, that the seagtgttcctgatgtatatyccgttggtgatgttgctacgttccctttgaaattgtacaat quence of the carboxyl-terminal end of the deduced amino acid 901 """".+"""".+"-----""------""+".".."+""..".+ S V P D V Y A V G D V A T F P L K L Y N sequence of pea MDAR Ser-Lys-Ile bears resemblance to the gacgtgayaagagttgaacacgttgatcatgctcgcaaatcagctgagcaggctgcaaag typical peroxisomal targetting sequence, althoughthe car961 """"~+""""~+"--"""+"...""+.....""+"....."+ D V R R V E H V D H A R K S A E Q A A K boxyl-terminal amino acid is usually leucine rather than isoleucine (23). Future studies will be aimed at subcellular localgccatctttgcagcagatgtagyaaaatcagttgaagagtatgattaccttccatacttc 1021 """".+"""".+"--""-+"+."".".*.....""+"."....+ ization of the isoform corresponding to this MDAR cDNA. A I F A A D V G K S V E E Y D Y L P Y F Comparison with protein sequences identified in a search of tattcccgttcytttgatctgtcctggcaattctatgqcgacaatgttgytgagacagtg the GenBank data base revealed that pea MDAR has signifi1081 " " " " ~ + " " " " ~ + ~ " " " ~ . + ~ " ~ ~ ~ ~ " + ~ ~ ~ " ~ ~ " + ~ ~ " ~ " ~ . + Y S R S F D L S W Q F Y G D N V G E T V cant homology with amino acid sequences of bacterial flavin ctatttgyagacaacgatcctgcatcatcaaagcctaaatttgggacatactggattaaa disulfideoxidoreductases. The highest scoring bacterial oxi1141 """"~+"""".+-"""...~..""-+..""".+".."".+ L F G D N D P A S S K P K F G T Y W I K doreductases were putidaredoxin reductase (24), rubridoxinreductase (251, terpredoxin reductase (261, ferredoxin reductase gaagygaaagttgttggggcctttttggagggtggaactcctgatgagaataaagctatt 1201 """"~+""""~+--------"---""-+..."."~+."..""+ (27), and toluene reductase (28) from Pseudomonas species, E G K V V G A F L E G G T P D E N K A I NADH oxidase from Enterococcus faecalis (29), trypanothione qccaaagttgcaagagccaagcctgcagtggaggatgtgaatcaacttycagaggaaggc reductase from !Oypanosoma brucei (30),glutathione reductase 1261 """"~+""""~+"---"-"----""+"""".+"."""+ A K V A R A K P A V E D V N Q L A E E G from Streptococcus thermophilus, and nitrite reductase from ctttcttttgcaagtaaaatttaatgatattcttgggaaggataaattgtttggtgattc Aspergillus nidulans (31). The amino acid sequences of the 1321 """"~~""""~+"-------'-""----t"""".+"."""+ L S F A S K I ' above mentioned enzymes rangedfrom 23 to 29% identity and 49 to 54% similarity, when compared with the pea MDAR decacagaacatattcgtattggtttctttttgctataggttatatcttayggaacttgtgt 1381 """".+""""~+--------"""----f""..".+".".."+ duced amino acid sequence. ttctttgttttaaatatgattgtgtgggatatgctttacgctgtgtactgaaccaaaaaa Close examination of the deduced amino acid sequence of 1441 "~~"".+""""~+~""."~+."...".+.~....."+"."....+ MDAR shows three putative regions that could be involved in taaaatcccttaqaaataataayaccttattygcaaaaaaaaaaaaaaaaaaaaaaaaaa binding FAD and NAD(P)H. An 11-amino acid consensus se1501 """""""""~+"-------f---------i".".."+"""...+ quence rule has been described (32) in which sequence motifs aaaaaaa 1561 - .- ...predicted to be that score as high as 9 out of 11amino acids are "core consensus" of the motif FIG.1. Nucleotide and deduced amino acid sequence of pea NAD(P)H bindingsites.The MDAR cDNA Nucleotides in bold lettering represent the typical eu- GXGXYGIA is found a t two distinct regions of the deduced karyotic translation initiation consensus surrounding the AUG initiation codon and the putative polyadenylation signal inthe 3"noncoding region. The underlined deduced amino acid sequences representputa- amino acids in boldlettering deviatefrom the consensus sequences tive motifs involved in FAD and NAD(P)H binding. The underlined discussed under "Results and Discussion." A

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amino acid sequence (Fig. 3A). The motif seen at the amino terminus is less certain to be involved in NAD(P)H binding, as the score for this motif is 8 out of 11, allowing nonconservative substitutions. A second motif implicated in FAD binding matches with the amino acid fingerprint found in otherflavincontaining enzymes (Fig. 3B). Crystal structures of some of these flavin enzymes have been resolved (33361, thusproving the involvement of the above described motif in FAD binding. The presence of these sequence motifs in the deduced amino acid sequence supports theidentification of the cloned cDNA as MDAR and reaffirms the involvement of NAD(P)H and FAD in the catalytic activity. Expression of MDAR in E. coli-The open reading frame encoding pea MDAR was fused to the3’ end of the E. coli MBP coding sequence to facilitate purification of pea MDAR. The fusion protein was found to be somewhat unstable in E. coli XLlB (Stratagene) because of partial proteolysis in vivo; thus expression was optimized in the protease-deficient E. coli 745 host strain. The expressed fusion proteinwas purified by affinity chromatography and subjected to cleavage by factor Xa. This protease recognizes the aminoacid sequence Ile-Asp-GlyAsn a t t hjunction e of the MBP-MDARfusion, releasing MDAR with an authentic amino-terminaldomain. The fusion protein was completely cleaved only after incubation for 48 h at room temperature, leading to the loss ofMDAR activity. Purified

MDAR from cucumber (11)and soybean (12) hasbeen found to bethermolabile. The factor Xa-digested fusion proteinwas separated by SDS-polyacrylamide gel electrophoresis, resulting in two major polypeptides of 42 and 47 kDa corresponding to MBP and MDAR, respectively (Fig. 4). However, because of persistent partial proteolysis in vivo even under theoptimized conditions of expression, a few minor proteolytic products in addition to the two major products were also observed. of MBPsMDAR Fusion Enzymatic Characterization Protein-Activity assays performed on the MBP.MDAR fusion protein, purifiedfrom the E. coli 745 crude extract,showed that the peaMDAR fusion has enzymatic properties very similar to cucumber (11)and soybean MDAR (12) (Table 11). Control assays with the MBPSlacZ fusion protein, purifiedfrom E. coli 745 transformedwithintact pMAL-c2, showed no background MDAR activity with eachof the electron acceptors usedin this study. The apparentK , values for NADH, NAD(P)H, and MDA are 5.3, 21.5, and 5.7 p ~respectively. , The V,, values are 312 pmol of NADP oxidizedmg of proteidmin and 40.9 pmol of NADPH oxidizedmg of proteidmin. Thus, both NADH and NADPH can serveas electron donors to pea MDAR in contrast to fungal MDAR, which lacks the ability to use NADPH (13). The enzyme has a broad pH optimum between7.5 and 9.5.The cloned MDAR activity is inhibited by 0.5 m~ p-chloromercuribenzoate and 0.1m~ 5,5‘dithio-bis(2-nitrobenzoicacid), indic-

1

A Protein Sequence (coenzyme)

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Aminofingerprint acid

GXGXXA/G

M)AR(NADH/NADPH)(P.sativum) MDAR(NADH/NADPH)(P.sativu~)

GG G V S A G GG Y I G G A G Y I A

11 169 174

G S S V V A

277

GGGFI S

195

Glutathionereductase (NADPH)(E.coli) Mercuric reductase (NADPH)(P.aeruginosa) Trypanothione reductase (NADPH)(T.congolense)

cDNA

Monodehydroascorbate PeaReductase

--

97.4

--

66.2

B Protein

Sequence

Location .

Amino fingerprint acid

T X X X X I Y I I G D M vwvv AFAA L L

MOAR (P.sativum) D i hydro1 ipoami de dehydrogenase (A.vinelandii) Glutathione reductase (€.coli) Glutathione reductase (human) Mercuric reductase (P.aeruginosa) p-Hydroxybenzoate

TS V P

D

286-296

S V P G V Y A IG D

308-318

T N I E G IY A V G D

293-303

T N V K G IY A V G D

321-331

T S N P N I Y AA

393-403

T

D

V Y A V G

GD

-- 45.0

. P

r)

;

- - 31.0

FIG.4. Coomassie Blue-stained SDS-polyacrylamide gel electrophoretogram of the MBP.MDAR fusion protein expressed in FIG.3. Comparison of putative NAD(P)Hand FAD binding mo- E. coli. Lane 1, 50 pg of crude extract from E. coli with recombinant tifs of pea ”Ft with similar motifs in other oxidoreductases. pMAL-c2. Lane 2, 50 pg of crude extractfrom E. coli with nonrecombiPanel A, the depicted motif has been found to be involved in binding to nant pMAL-c2. Lane 3, 5 pg of fusion protein following elution from NAD(P)H in oxidoreductases for which crystal structures have been amylose affinitycolumn. Lane 4.10 pg of fusion proteinfollowing factor resolved (refer to “Results and Discussion”). T w o distinct motifs, oneat Xa digestion. Lane 5 , 5 pg of purified pea MDAR after electroelutionof the amino terminus and one closer to the carboxyl terminus, are the present 47-kDa polypeptide. Lane 6,molecular mass markers (Bio-Rad): in theMDAR deduced amino acid sequence of pea with respective scores rabbit muscle phosphorylaseb (97.4 kDa), bovine serum albumin (66.2 of 8/11 and 9/11. Panel B, a similarcomDarison indicates the presence of kDa),chickenovalbumin(45kDa), and bovine carbonic anhydrase an FAD bindingdomain.nearthe second NAD(P)H binding’domain. (31 ma).

hydroxylase(P.fluorescens)

M Q GH

R LF

LA G

D

276-286

Pea Monodehydroascorbate Reductase cDNA

31133

TABLE I1 Comparison of enzymatic propertiesof pea MBPaMDAR fusion protein withpurified MDAR from soybean a n d cucumber MBP.MDAR fusion protein

Enzymatic property

312 NADH donor as electron 40.9 NADPH donor as electron MDA donor a s electron DCPIP acceptor a s electron FeCN acceptor as electron o/o Inhibition by 0.5 mM p-chloromercuribenzoate 100 0.1 mM 5-5' dithiobisnitrobenzoate 93 pH

K."

V."*.

pmollmglmin

PM

pmollmglmin

23 NDh 331

26.6

30

Cucumber MDAR (11)

Soybean MDAR (12)

V"2

5.3 21.5 5.7 52 ND

288 150 ND ND 425 30.5 203

K", PM

12

V,",

K",

ymollmglmin

p.M

5.6 150 7.0 ND ND

ND

4.6 1.4

25 94 96 7.0-9.0

100

ND 7.5-9.5

200

8.0-9.0

Note: K, in PM;V,, in pmol NAD(P)H oxidizedmg proteinlmin. * ND, not determined; DCPIP, dichlorophenolindophenol;FeCN, ferricyanide.

1

mAR

I

2

3

4

5

6

Acknowledgments-We thank Dr. J. Stephen Gantt for generously providing the pealeaf hgtll cDNA library, Drs. PeterKahnand Theodore Chase for helpful discussions, and Dawn Howell for help in preparing photographs. REFERENCES

1. Foyer, C. H., and Halliwell, B. (1976)Planta (Heidelb.) 133,21-25 2. Nakano, Y., and Asada, K. (1981)Plant Cell Physiol. 22,867-880 3. Yamazaki, I., and Piette, L. H. (1961)Biochim. Biophys. Acta 50,62-69 4. Cabelli, D. E., and Bielski, B. H.J. (1983)J. Phys. Chem. 87, 1809-1812 5. Packer, J. E.. Slater, T. F., and Willson, R. L. (1979)Nature 278, 737-738 6. Scarpa, M., Strevanato, R., Viglino, P., and Rigo, A. (1983)J. Biol. Chem. 258, FIG.5. Northern blot analysisof total RNA from various pea 6695-6697 tissues. A blot containing30 pg of total RNA from each tissue source 7. Morre, D. J., Navas, P., Penel, C., and Castillo, F. J. (1986)Protoplasma 133, was probed with the radiolabeled pea MDAR cDNA, exposed to x-ray 195-197 film, and probed subsequently with a n 18 S rRNA to assess RNA loadT. J., andLuster, D.G. (1991)in OxidationatthePlasma ing. Lane 1,light-grown pea leaves; lane 2, dark-grown pea leaves; lane 8. Buckhout, Membrane: Relation to Growth and Ttansport (Morre, D. J., Frederick, L. 3, young leaves; lane 4 , old leaves; lane 5, flowers; lane 6, roots. C., and Low, H. E.,eds) vol. 2,pp. 21-33,CRC Press, Boca Raton, FL Dipierro, S., and Borraccino, G . (1981)FEBS Lett. 125,242-244 9. Arrigoni, 0.. ative of the involvement of a thiol group in MDAR activity. 10. Yamanuchi, N., Yamawaki, K., and Ueda,Y. (1984)J. Jpn. Soc. Hortic. Sci. 53, 347-353 Dichlorophenolindophenol and ferricyanide can serve as alter- 11. Hossain, M.A., and Asada, K. (1985)J. B i d . Chem. 260, 12920-12926 nate electron acceptors; the former is a much more effective 12. Dalton, D. A,, Langeberg, L., and Robbins, M. (1993)Arch. Biochem. Biophys. 292,281-286 electron acceptor than the latter. The fusion protein was quite 13. Schulze, H.U., Schott, H. H., and Staudinger, Hj. (1972)Hoppe-Seyler5 Z. stable and did not lose significant activity over a period of 1 Physiol. Chem. 353, 1931-1942 month when storedat 4 "C in thepresence of 10 mM P-mercap- 14. Meister, A. (1994)J . Biol. Chem. 269,9397-9340 15. Sano, S., and Asada, K. (1992)in Research in Photosynthesis (Murata, N., ed) toethanol. vol. 4, pp. 533-536, Kluwer Academic Publishers, Dordrecht, The Analysis of Pea MDAR Steady-stateDanscript LmelsNetherlands MDAR expression was examined by Northern hybridization 16. Benton, W. D., and Davis, R.W. (1977)Science 196,180-182 F., Nicklen, S., and Coulson, R. (1977)Proc. Natl. Acad. Sci.U.S. A. 74, analysis of RNA isolated from roots, youngand old leaves, and 17. Sanger 5463-5467 flowers, as well as from leaf tissue from 10-day-old seedlings 18. Riggs, P. (1990)in Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Smith, J. A,, Seidman, J. G., and grown in complete darkness or in thegreenhouse. The level of Wiley Interscience, New York Struhl, K., eds) pp. 16.6.1.-16.6.12, pea MDAR transcripts monitored at high stringency wasfound 19. Bradford, M. M. ( 1976)Anal. Biochem. 72,248-254 to be fairlylow in all tissuesexamined (Fig. 5) relative to other 20. Laemmli, U. K., and Fawe, M. (1973)J. Mol. Biol. 80,575-599 transcripts encoding antioxidant enzymes as measured by 21. Chomczynski, P., and Sacchi, N. (1987)Anal. Riochem. 162, 156-159 22. Mittler, R., and Zilinskas, B. A. (1994)Plant J. 5,397405 Northern hybridization analysis.2 After normalization of the 23. Gould, S. J., Keller, G.-A,, and Subramani, S. (1987)J. Cell Biol. 105, 2923Northern blot data with respect to the 18 S rRNA signal, it was 2931 J. A,, Lorence, M. C., and Amameh, B. (1990)J. Bid. Chem. 265, found that flowers had a slightly higher(-2.5-fold) level of pea 24. Peterson, 6066-6073 MDAR transcript relative to other tissues. Likewise, there was 25. Eggink, G., Engel, H., Vriend, G., Terpstra, P., and Witholt, B. (1990)J. Mol. B i d . 212, 135-142 a slightly higher level of MDAR transcript (--1.7-fold) in leaf J. A., Lu, J.-Y., Geisselsoder, J., Garaham-Lorence,S., Carmona, C., tissue from dark-grownpeaseedlings relative to the light- 26. Peterson, Witney, E , and Lorence, M.C. (1992)J. Biol. Chem. 267,14193-14203 grown plants. 27. Tan, H.-M., Tang, H.-Y., Joannou, C., Wahab, N. H., and Mason, J. A. (1993) Gene (Amst.) 130,3349 Concluding Remarks-In conclusion, we have demonstrated 28. Zylstra, G. J., and Gibson, D. T.(1989)J. Biol. Chem. 264,14940-14946 that the cloned cDNA encodes the ascorbate-regeneratingen- 29. Ross, R. P., and Claiborne,A. (1992)J. Mol. Biol. 227, 658-671 zyme, MDAR, either a cytosolic or peroxisomal isozyme. Pea 30. Abagye-Kwarteng, T., Smith, IC, and Fairlamb,A. H. (1992)Mol. Microbiol. 6, 3089-3099 MDAR shows high degree of homology in termsof amino acid 31. Johnstone, I. L., McCabe, P. C., Greeves, P.. Cole, G.E., Brow, M. A. D., Unkles, sequence with microbial flavinoxidoreductases and is exS. E., Clutterbuck, A. J., Kinghorn, J. R., Innis, M.A. (1990)Gene (Amsf.) 90, 181-192 pressed in a wide range of tissues. The availability of this 32. Wierenga, R. K., Terpstra, P., and Hol, W. G. J. (1986)J. Mol. B i d . 187, cloned cDNA should facilitate cloning of the mammalianhomo101-107 logue as well as molecular analysis of the regulation of the 33. Krauth-Siegel, R. L., Sticherling, C., Jost, I., Walsh, C. T., Pai, E. F., Kabasch, W., and Lantwin, C. B. (1993)FEBS Lett. 317, 105-108 expression of this enzyme during development and inresponse 34. Schiering, N.. Kabasch, W., Moore, M. J., Distefano, M. D., Walsh, C. T., and to oxidative stress. Pai, E. F. (1991)Nature 352, 168-172 35. Mattevi, A,, Schierbeek, A. J., and Hol, W. G. J. (1991)J. Mol. B i d . 220, 975-994 36. Erlmler, U.,and Schulz,G.E. (199l)Proteins Struct.Funct. Genet. 9,174-179 B. A. Zilinskas, unpublished data.