Proc. Natl. Acad. Sci. USAVol. 91, pp. 8552-8556, August 1994Plant Biology

Expression cloning of a gibberellin 20-oxidase, a multifunctionalenzyme involved in gibberellin biosynthesis

(2-oxoglutarate-dependent dloxygenase/in vio tanslaton/cDNA doning/deduced amino add sequence)

THEODOR LANGE*, PETER HEDDENt, AND JAN E. GRAEBE**Pflanzenphysiologisches Institut und Botanischer Garten der Universitit Gottingen, Untere Karspfile 2, D-37073 Gdttingen, Germany; and tDepartment ofAgricultural Science, University of Bristol, Institute of Arable Crops Research, Long Ashton Research Station, Bristol BS18 9AF, United Kingdom

Communicated by J. MacMillan, May 10, 1994 (received for review June 16, 1993)

ABSTRACT In the biosynthetic pathway to the gibberel-lins (GAs), carbon-20 is removed by oxidation to give theC19-GAs, which include the biologically active plant hormones.We report the isolation of a cDNA clone encoding a GA20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase(20-hydroxylating, oxidizing) EC 1.14.11.-] by screening acDNA library from developing cotyledons of pumpkin (Cucur-bita maxima L.) for expression of this enzyme. When mRNAfrom either the cotyledons or the endosperm was translated invitro using rabbit reticulocyte lysates, the products containedGA12 20-oxidase activity. A polyclonal antiserum was raisedagainst the amino acid sequence ofa peptide released by trypticdigestion of purified GA 20-oxidase from the endosperm. AcDNA expression library in Agtll was prepared from cotyledonmRNA and screened with the antiserum. The identity ofpositive clones was confirmed by the demonstration of GA1220-oxidase activity in single bacteriophage plaques. Recombi-nant protein from a selected clone catalyzed the three-stepconversions ofGA12 to GA2s and ofGA53 to GA17, as well as theformation of the C,,-GAs, GA1, GA,, and GA20, from theirrespective aldehyde precursors, GA23, GA2,4, and GA19. Thenucleotide sequence of the cDNA insert contains an openreading frame of 1158 nt encoding a protein of 386 amino acidresidues. The predicted Mr (43,321) and pI (5.3) are similar tothose determined experimentally for the native GA 20-oxidase.Furthermore, the derived amino acid sequence includes se-quences obtained from the N terminus and two tryptic peptidesfrom the native enzyme. It also contains regions that are highlyconserved in a group of non-heme Fe-containing dioxygenases.

The gibberellins (GAs) form a large group of diterpenoidnatural products. Certain GAs function as hormones inplants, controlling many aspects of development, includingstem extension, fruit set, and seed germination (1). The latersteps of the GA biosynthetic pathway are catalyzed bysoluble 2-oxoglutarate-dependent dioxygenases, which oxi-dize the GA skeleton at the 20, 3,8, and 2p3 positions (see Fig.1) (2). Oxidation at carbon-20 leads to the loss of this atomand to the formation of the physiologically active C19 GAs.Immature seeds of Cucurbita maxima are a rich source of

GA-biosynthetic enzymes, and cell-free preparations fromseed tissues of this species have proved of considerable valuein elucidating GA-metabolic pathways (3, 4) and in charac-terizing the enzymes involved (5). Recently, GA12 20-oxidase[gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing) EC 1.14.11.-] has been purified toapparent homogeneity from the endosperm/nucellus andshown to catalyze the conversion of GA12 to GA25 and ofGA53 to GA17 and GA20 (see Fig. 1) (6). In this paper we reportthe isolation of a cDNA encoding this enzymet and show that

it belongs to a group of non-heme Fe-containing dioxygen-ases, based on its derived amino acid sequence.

MATERIALS AND METHODSPoly(A)+ RNA Isolation, in vitro Translation, and 20-Oxi-

dase Assay. Cotyledons and endosperm were harvested frompumpkins (C. maxima L. cv. Riesenmelone, gelb genetzt) asdescribed (3, 4). Poly(A)+ RNA was isolated from thesetissues using the Fast Track mRNA isolation kit (Invitrogen).The yield of poly(A)I RNA from cotyledons (17.4 ,ug/g offresh weight) was much higher than that from endosperm(0.75 pg/g offresh weight). In vitro translation ofmRNA wasdone with rabbit reticulocyte lysates (Boehringer Mannheim)using the manufacturer's recommended conditions, exceptthat leucine and methionine were at 12.5 ,uM. After incubat-ing for 2 hr at 300C the mixtures (50 .A) were supplementedwith dioxygenase cofactors (4 mM 2-oxoglutarate/0.5 mMFeSO4/4 mM ascorbate/and catalase at 1 mg/ml) and[14C]GA12 (15,000 dpm; 37.9 pmol) added in 5 ;4 and incu-bated for a further 3 hr at 300C. The reaction was terminatedby adding acetic acid (5 pl) and acetone (1 ml twice) andpelleting the protein and nucleic acids by centrifugation at5000 x g for 5 min. The supernatant was taken to dryness invacuo, and the metabolites were analyzed as described (3).cDNA Library Construction. An oligo(dT)-primed cDNA

expression library was prepared in Agt11 from cotyledonpoly(A)+ RNA (5 pg) using commercial kits (cDNA synthesissystem plus and cDNA cloning system - Agtll; Amersham).The library contained 7 x 104 plaque-forming units (pfu), ofwhich 60% of clones were shown to contain inserts >800 bpby agarose gel electrophoresis ofPCR products using Agtll-specific primers (7). The library was amplified in Escherichiacoli strain Y1090hsdR, according to the manufacturer's in-structions.

Production of Antibodies. The purification of GA12 20-oxidase and the determination of amino acid sequences forthe N terminus and several tryptic peptides are describedelsewhere (6). A peptide (VFGGSDESK) was synthesized onthe basis of one such sequence and used for the preparationof antibodies. The synthetic peptide (2 mg) was linkedcovalently to hemacyanin (2 mg) with glutaraldehyde (0.5ml), and the conjugate (400 pg in Freund's adjuvent) was usedto elicit polyclonal antibodies in a rabbit by s.c. injection,repeated six times with 200 pg of conjugate at 3-weekintervals. The IgG fraction was purified from the antiserumon a protein A column and, after concentration, stored at-400C.Screening the Library with an Antibody and Enzyme Ac-

tivity. Two 90-mm plates each containing 3600 pfu wereimmunoscreened (8) with the peptide polyclonal antibody (1

Abbreviations: GA, gibberellin; pfu, plaque-forming units.tThe sequence of the cDNA encoding the GA 20-oxidase has beendeposited in the GenBank data base (accession no. X73314).

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3'

GA12:Ri-H GA I i(opsnlstonm):R I-HGA53:Rl-OH GA44(open lkorn):Ri-OH

GA24: Ri-H,R 2-HGA19: R g-OH,R 2=1GA23: Ri-OHR 2s

RiHOOO

R 2'eH OOH-# COOH

GA25:Ri-HR 2-HGA17:R1-OH,R 2-HGA29:RI-OH,R g-OH

H R2DO-H GA9 :R1H,R2-H

GA2:Ri-OH,R 2-HGA1 :R1OH,R2-OH

FIG. 1. Structures and metabolic relationship of GAs discussed in the text.

pzg/ml) and an alkaline phosphatase-conjugated anti-rabbitIgG second antibody. Nine positive plaques (designatedkA76-84) were obtained, and these, as well as one negativeplaque as control, were each replated at 50 pfu per plate andrescreened. Two positive plaques from each replating wereassayed for GA12 20-oxidase activity by incubating agar plugs(=25 jil) in Eppendorf tubes containing [14C]GA12 (15,000dpm, 37.9 pmol) and cofactors as given above in 50 mMTrisHC1, pH 7.5/100 mM NaCl/10 mM MgSO4/0.010%gelatin (25 p4). Incubations were for 6 hr, replenishing thecofactors every 2 hr. Products were recovered after centrif-ugation (15,000 x g for 2 min) and separated by reverse-phaseHPLC as described (3). The size of inserts in the positivebacteriophages was determined by PCR (7).

Preparation and Characterization of Recombinant Protein.A lysogen was prepared from bacteriophage AA79 in a 250-mlculture as described by Sambrook et al. (8). After lysis andcentrifugation different aliquots of the supernatant (=1 ml)were incubated in a total volume of 265 Al with [14C]GA12 or[14C]GA53 (100,000 dpm, 255 pmol) and cofactors as de-scribed above for 4 hr at 30'C; fresh cofactors were added in12.5 Al after 2 hr. Also 14C-labeled GA24, GA19, and GA23(200,000 dpm, 510 pmol) were incubated separately with 250A4 of the lysate for 6 hr, replenishing the cofactors every 2 hr.Products from each incubation were extracted and analyzedby HPLC radiocounting. Fractions containing radioactivitywith the same retention time were combined from each seriesof incubations, and the identity of radiolabeled products wasconfirmed by comparison of their mass spectra, determinedby full-scan GC-MS, with spectra of unlabeled standards (9)as described in ref. 4. To determine the cofactor requirementsofthe recombinant protein, the lysate (150 /4) was gel-filteredby using a NAP-5 column (Pharmacia) and eluted in 1 ml of100mM Tris-HCl, pH 7.0 at 30'C, before incubation. Aliquotsof the eluate (5 ILI) were incubated with different cofactorcombinations and [14C]GA12 (20,000 dpm, 51.1 pmol) in atotal volume of 100 /4 for 2 hr at 30TC. Products wereextracted and analyzed by HPLC with on-line radiocounting(3).

Subcloning and DNA Sequencing. A 400-ml culture of E.coli strain YlO9OhsdR was infected at high multiplicity (_1013pfu) with phages of clone AA79 as described by Sambrook etal. (8). The bacteriophage DNA was purified using a A phagepurification kit (Qiagen, Chatsworth, CA). After release ofthe insert with BamHI it was subcloned into the BamHI siteofpUC18, and transformants were selected with E. coli strainXLlblue. Plasmid DNA was isolated from one transformant(pB9) using a Plasmid Midi kit (Qiagen) and sequenced on

both strands by the dideoxynucleotide chain-terminationmethod (10).Sequence Aliment. The amino acid-sequence alignment

was made using the PILEUP program from the GeneticsComputer Group package run on a VAX computer.

RESULTSIn Vitro Translation of Poly(A)+ RNA. After translation of

poly(A)+ RNA from developing cotyledons or endosperm ofpumpkin in rabbit reticulocyte lysates, the products con-verted [14C]GA12 to [14C]GA15, as identified by HPLC, whenthey were incubated with the appropriate cofactors (Figs. 1,2). This result demonstrated that the 20-oxidase gene wasbeing expressed in both cotyledons and endosperm. The

a

C! ~~~~~~II

d 11

0 10 20 30Retention time (min)

FIG. 2. GA 20-oxidase activity in products from the in vitrotranslation of C. maxima poly(A)+ RNA. [14C]GA12 and dioxygenasecofactors were incubated with translation products produced byusing either rabbit reticulocyte lysate and tobacco mosaic virus RNA(1 jg) (a), cotyledon mRNA (1 ug) in the absence of the translationsystem (b), rabbit reticulocyte lysate and mRNA from C. maximacotyledons (1 jg) (c), or from endosperm (0.5 pg) (d). On the basisof HPLC retention times the peaks correspond to GA12 (I) and GA15(II).

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0)-

0

8 b

0 10 20 30Retention time (min)

FIG. 3. GA 20-oxidase activity in a recombinant bacteriophageplaque. [14C]GA12 and dioxygenase cofactors were incubated with25-/4 agar plugs containing plaques of single recombinant Agtllbacteriophage that were not recognized (a) or were recognized (b) byan anti-GA 20-oxidase antibody. On the basis of HPLC retentiontimes the peaks correspond to GA12 (I), GA1s (II), and GA24 and GA25(III).

activity was higher in the translation products derived fromthe cotyledon mRNA. No activity was detected after incu-bation of the reticulocyte lysate with tobacco mosaic virusRNA or of the cotyledon mRNA without the translationsystem.

Isolation ofcDNA Clones. Antibodies were raised against asynthetic peptide (VFGGSDESK), the sequence of whichwas based on the sequence of a peptide resulting from trypticdigestion of purified GA12 20-oxidase from C. maxima en-dosperm (6). These antibodies were used to immunoscreen anamplified Agtll expression library, prepared from cotyledonpoly(A)+ RNA. Nine positive plaques from 7200 recombi-nants (0.13%), after purification, were shown to convert[14C]GA12 to radiolabeled GA15, GA24, and GA25, as identi-fied by HPLC, when incubated with the substrate andcofactors (Fig. 3). Negative plaques contained no enzymeactivity (Fig. 3). There was complete correlation betweenantibody recognition by bacteriophage plaques and theirability to oxidize GA12. The size of the inserts in the positiveplaques were all -.1400 bp.

In a separate experiment three plaques of 3600 (0.08%)from an unamplified library were found to be antibody-positive, indicating an abundance of 0.5% of potential GA20-oxidase clones.

Production of Lysogens and Properties of the RecombinantProtein. We examined the catalytic properties of the recom-binant protein using a lysogen ofAA79 (Table 1). The capacityof the protein to oxidize [14C]GA12 was absolutely dependenton the addition of 2-oxoglutaric acid and was reduced by 67and S7%, respectively, when Fe2+ or ascorbic acid was

omitted. The substrate specificity of the protein was alsodetermined (Tables 2-4). When [14C]GA12 was incubatedwith increased volumes oflysate, the carbon-20 methyl group

Table 1. Cofactor requirements for the metabolism of [14C]GA12by cell lysates from E. coli lysogenized with AA79

Products after HPLC, %

Conditions GA12 GA15 GA24 GA25Pull cofactors 18 68 13 1No cofactors 100Without 2-oxoglutarate 100Without ascorbate 89 11Without FeSO4 73 27

Table 2. Dependency of [14C]GA12 metabolism by E. colilysogenized with AA79 on cell lysate volume

Products, % by HPLC

Lysate, /4 GA12 GA1s GA24/GA25*1.0 48 49 32.0 10 70 203.9 3 14 847.8 4 4 92t

All products were identified by comparison of their mass spectrawith published spectra (9): GA12 mass/charge (percentage relativeabundance)-368(2), 366(1.5), 360(0.7), 336(33), 334(19), 328(8),308(100), 306(57), 300(27), 247(23), 245(13), 241(6); GA1s-352(29),350(14), 344(15), 320(29), 318(16), 312(8), 290(55), 288(24), 284(17),245(100), 243(51), 239(23); GA24-350(51), 348(20), 342(15), 322(92),320(67), 318(67), 316(23), 314(45), 310(8), 294(55), 293(58), 290(61),288(27), 286(29), 232(84), 231(100), 230(42), 229(39), 226(27),225(25); GA2s-380(37), 378(20), 372(7), 320(100), 318(48), 312(26),292(92), 290(75), 284(27), 231(47), 229(20), 225(10).*Not resolved by HPLC.fShown by full-scan GC-MS to contain only [14C]GA2s.

was oxidized progressively to the alcohol, aldehyde, andcarboxylic acid to give, respectively, radiolabeled GA15,GA24, and GA2s as products (Table 2). The corresponding13-hydroxy GA products (GA44, GA19, and GA17) were alsoobtained, although in lower yields, when the lysate wasincubated with [14C]GA53 (Table 3). These properties corre-spond exactly to those of the native 20-oxidase isolated fromC. maxima endosperm (6). In a comparison of the aldehydesubstrates GA24 (nonhydroxylated), GA19 (monohydroxy-lated), and GA23 (dihydroxylated), the yield of the corre-sponding tricarboxylic acids decreased with increased hy-droxylation of the substrate (Table 4). In addition, thecorresponding C19-GA products (GA9, GA20, and GA1),which are formed by loss of carbon-20 as C02 (11), wereobtained in low yield. The identities of all products listed inTables 2-4, except for GA20 and GA24, were confirmed byfull-scan GC-MS. The products had the same specific radio-activity as the substrates and were, therefore, undiluted byendogenous GAs. This result confirms that these compoundsdo not occur naturally in E. coli.Suboning and Sequencing of cDNA Clone. The cDNA

insert from a purified plaque (clone A79) was subcloned intopUC18 after excision withBamHI. The sequence ofthe insertcontained an open reading frame of 1158 nt encoding aproteinof Mr 43,321. The N-terminal amino acid sequence of nativeGA 20-oxidase purified from C. maxima endosperm(LNEEMKGEYRPPFGG) and the sequences of two trypticpeptides prepared from this protein (GEYRPPFGGSDESK

Table 3. Dependency of [14C]GAs3 metabolism by E. colilysogenized with AA79 on cell lysate volume

Products, % by HPLCLysate, pl GA53 GA" GA19 GA17 GA20

7.8 42 58 0 0 015.6 13 79 8 0 131.3 1 69 25 5 162.5 0 49 39 10 2125 0 29 46 23 2250 0 16 43 38 2

All products, except for GA2o, were identified by comparison oftheir mass spectra with published spectra (9): GAs3 mass/chage(percentage relative abundance)-456(76), 454(42), 448(28),424(22), 422(13), 416(10), 395(39), 393(18), 389(10), 209(100),207(50); GA44O440(62), 438(34), 432(29), 379(19), 377(8), 373(8),240(38), 238(20), 209(100), 207(50); GA19--442(100), 440(37),434(33), 410(35), 408(13), 402(9), 381(70), 380(79), 379(23), 378(18),375(20), 374(25); GA17-500(23), 498(11), 492(11), 468(19), 466(15),460(8), 409(21), 407(11), 401(13), 210(100), 208(45).

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Table 4. Metabolism of 20-oxo[V4C]GAs by cell lysates from E.coli lysogenized with AA79

Products, % by HPLC

Substrate GA7s/GA9* GA17 GA20 GA28 GA1GA24 96GA19 49 0.9GA23 3.4 0.4

All products, except for GA23, were identified by comparison oftheir mass spectra with published spectra (9): GA2s mass/charge(percentage relative abundance)-380(38), 278(19), 372(34),320(91), 318(47), 312(100), 292(76), 290(68), 284(79), 231(49),229(23), 225(44); GA9-306(100), 298(31), 276(54), 270(22), 227(63)(weak spectrum); GAl?-500(27), 498(11), 492(14), 468(18), 466(12),460(11), 409(16), 407(10), 401(8), 210(100), 208(31); GA20o.426(100), 424(57), 418(56), 381(60), 379(25), 375(27), 365(20), 363(9),359(7); GAi-514(100), 512(54), 506(54), 454(18), 453(17), 452(10),451(13), 448(7), 447(7), 382(21), 376(12).*Not resolved by HPLC; the proportion of GA9 was estimated byGC-MS to be <1%.

and NFFEDNDSILR) (6) are present in the predicted se-quence, starting at amino acid positions 16, 22, and 214,respectively (Fig. 4). The sequence against which the anti-body was raised, with one change (proline for valine), formsthe C terminus of one of the tryptic peptides (amino acids27-35 ofthe predicted sequence). The discrepancy was foundsubsequently to be due to the presence of an impurity in thispeptide. The theoretical molecular mass and pI (5.3) of theencoded protein are similar to those determined for thepurified native enzyme (Mr 44,000 and pI 5.4) by SDS/PAGEand isoelectric focusing electrophoresis, respectively (6).Comparison of the derived amino acid sequence with thosefrom other plant dioxygenase genes (Fig. 4) shows highlyconserved regions, although overall amino acid identity isonly 25-30%/o (17). The conserved sequences include three

histidine-containing motifs, which, in the GA 20-oxidase,contain His-95, His-243, and His-298.

DISCUSSIONWe have presented evidence that a cDNA derived fromimmature pumpkin cotyledons encodes a GA 20-oxidase.Lysogenized E. coli expressing the cDNA as a fusion proteincontainedGA 20-oxidase activity. The cofactor requirementsand substrate specificity of the expressed enzyme are iden-tical with those of GA 20-oxidase purified from pumpkinendosperm. Furthermore, the Mr and pl, calculated on thebasis of the amino acid sequence encoded by the cDNA, arealso similar to those measured for the native enzyme. Inaddition, the derived sequence contains sequences shown tobe present in the native enzyme. The peptide sequence usedto prepare the antiserum for screening the cDNA library waspresent in the theoretical sequence starting at residue 27 anddoes not include sequences conserved in other plant dioxy-genases. Thus, the antiserum should be specific for the20-oxidase. Because this peptide sequence is located close tothe N terminus, cDNA clones detected with this antiserumwould all be close to full-length and, as we have demon-strated, would encode functional proteins. The success ofthefunctional assay, particularly the demonstration of enzymeactivity in individual bacteriophage plaques, suggests itspotential utility as a screen of the library in the absence ofanantibody.A remarkably high abundance ofmRNA for the 20-oxidase

in both endosperm and cotyledons was indicated by thedetection of enzyme activity in the in vitro translation prod-ucts. The cotyledons proved a much richer source ofpoly(A)+ RNA than did the endosperm and were used toprepare the cDNA library. The abundance of 20-oxidaseclones in the unamplified library can be estimated as 0.5%and may be in excess of this because the antibody used to

200x . MALNGKVAT K AP S NL NE EMKG E YR P P FGGS DE S KVP EDaI WS E K F E|S EL L P.. V L DVOTD E K F M S GDF S YV EBAT RLV D 81E8 MESPRVES YDEMSKLEAFDDTEAGVEGLVDSGITK.VPQI VLPPKDRO KC.E T FY DFQVGI.DEDPIRKHR IVDKVR 81Efe . . . . . . . . . . . . . . . . . . . . . M E N F N E N L N G D Z R A T M E M I R 25

HY06h.........................MTVNSKVE 3ALK[ED.VNV DQQ. .HLLVQQIT 52F3h..................... IPRVTPSTLTALAEETLQTSUIRD8DERPKVAYNQFSNEI S EGI .DDETGKRA ICDIV 64A2 M SSPLLQLPAARVKALSLSaLSAIPPIYVRPADERAGLGDAFDLARTSENDHTAPRIPVVD SPFLDS . SSQQQQRDDCVEAVR 84

2008o R VHIV DIHMWG RV ED CKMNEMT M[]LDVQRAERKVRES...YGYTNSFF G R FA S Z; L P[3KE.T.FSLRCVAAQNSS 162ES D2BKFQK 3I T5VLDRTLQG TRQUREQDN VBKQYYTRDTAK VVYTSNL DLYRSSVPAASWRDTIIFCYMAPNPP... 163

Efe DEN rF EL VWDTVZKLTQKGaX.?CM QRFh.KLVASK.....GLEA...VQAEVTDLDW .STFFLRELPTSN. 99

Kyo~h E5QDjLQI I

WL LETKIVCK AL A

K ZEFXP9GZAAKFZLPLRQKARLYVEGZQLSN YFLYWKDTLAHGCE 137

F3h EDXVQ D VDA VISQMTTFAEE AL P LRF DMHSaGEGGFIVSSE. .. LQGZVVQD R IVTYFSYPTRARD. 145S IDADVXHZAQ IaA LBDRLRAAGTA AL VQD ZAY.ANDPAAGRLQGYSSRLATNTCGQRE EDYLFHELVHPDGLAD 168

20ox AAHDYVLDTLGP8r7BHHGEAYQECGIALNZ GTKZVELaGLS ....... ,BRE F NFFEDNDSI.....LRL TKDE 235F8 ..L.Q.....LFQBTPCaZSLIDFSEDVY OFT a L . G ...... DRB LKDYMM .. DCFELF. . CSC P Q 228

Efe ...... IBQV DLDEDYRVYRDFAERLMDFADKR LIC N ....... EKIG LCNAFYGSKGPNFG.TTV s P .Ef D 169

Eyo6h PLDQDLVNWEXEKAEYRZVVAKYBSVVRK TERR a ... L D.Q.QGFDNZL gQIQMM

.

P 20973h.. . . . YsRY DUbGNWIAVTQEYBEELME ACK LDVIA .f..EEALTEA...CVDMDQE..VVV K X D 212A2 AL .... W AYPDYIAATRDFORRTRD ASBTAI SMGDLc2T;DRGDAEEALTTTTTRTAADDDLLLQLKI RE Q E 246

20ox V V L T P P TVsVoI R * P5se VCSDQ.. YS P NOEAFA I T TSBT IflEC II AVV8Y]S N A K LMF L C 317EfS fJT TZQ 'IarV I L D.MG a. .YVLKQXK VD P TGS8L I F QLS DEELBV AI5 NVY H ITC FG 310Ef IE LRA *AGOZIL F fDEs *LLEDEQ. . IDflPDNREBI LimQ EVIT KflKBVM VIAQTDOT N L BOYN 252

Eyo6h S T L SBG . .L j L Q .LP DQLIAVrDATA Q I[IT AFL T V r T EErEQa s I VVTDPTRD V I T, I a 292T3hT L LEI R PSOTI LL NQ GG TXDNaKT IT Q v xaTAF L nKEraS RU; PiNA Q)AVVISHNBS L I T Q N 296A2 AV V Z A 'VBALBrILBa. P VL . . EOAR VTARHEG(;Tr HVT AEIs I0S RIT S V L. GLV REAV I W YV CZ 328

200X DSH.0EVV.RAE VEKgBPRKEYPDYEWPMLLEHTQERYRPDCNTLEAF TWVQEGKALDTGSTITAPSAA 0r386Z8 ISPYQSBELYG IT LSEDNHPEYRATTVEDETBYLENROLDOTSALSRY I ......................... 363Efe DN.RAVIYPAU.BLIEzsQVYPxrVFDDYMK ......... LYAaLEFQP EPRFEAMXAME.ANVE..LVDQIASA316

Eyo6h ... YSCTIKDAKELNQDNDfPLYEPYSYSBKADIYLBDESDYDSGVEPY INV.. 34473h SAP .. AIVY LE.IRREg KSIMDEP ITFAKMYRRKMSEDLELARLEKQA ZQQLQAEVAAZKAKLESKPIKKILA.369AL2 PP. OSVLL.E LPP VTK ElHAR FTPRT FxQKLDR KL F K QQ K AKASKK DGGNGDDHHR PPPQTN . ..H.KP..P.T. 395

FIG. 4. Alignment of the derived GA 20-oxidase amino acid sequence (20ox) with other plant dioxygenase sequences: E8, a ripening-relatedgene of unknown function from tomato [Protein Identification Resource (PIR) 2:S01642] (12); Efe, ethylene-forming enzyme (1-aminocyclopropane-l-carboxylic acid oxidase) from ripening tomato fruit (PIR3:S16327) (13); Hyo6h, hyoscyamine 6p-hydroxylase fromHyoscyamus niger (PIR2:A40005) (14); F3h, flavanone 3p-hydroxylase from Petunia hybrida (PIR3:A42110) (15); and A2, gene product frommaize involved in anthocyanin biosynthesis (PIR3:S12043) (16). Identical residues in four or more sequences are shown in reverse script;conserved amino acids are indicated by light shading.

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screen the library would not detect short clones. The 20-oxidases expressed in cotyledons and endosperm must havevery similar, if not identical, amino acid sequences becausesequences determined from the endosperm protein are pres-ent in the deduced sequence from the cotyledon-derivedcDNA in regions not generally conserved in plant dioxyge-nases. It is likely, therefore, that the same gene is expressedin both tissues.The capacity of the recombinant enzyme to convert GA12

to GA25, and GA53 to GA17, indicates that a single enzymecatalyzes three consecutive oxidations at carbon-20 duringGA biosynthesis. Furthermore, the enzyme also catalyzesthe loss of carbon-20 from the aldehyde precursors and is,therefore, responsible for each step in the formation ofC19-GAs from the 10-methyl precursors. This conversion toC19-GA product is observed also with the purified 20-oxidase(6). There is no evidence that the loss of carbon-20 can occurnonenzymatically, nor does it occur in cultures of E. coli inwhich the pumpkin GA 20-oxidase is not expressed (data notshown). The aldehyde intermediate in the GA-biosyntheticpathway can be oxidized either to the carboxylic acid or togive the C19-GAs by loss of carbon-20. In contrast to mostother plant tissues, pumpkin seeds produce the C20 tricar-boxylic acids as major products-e.g., GA43 (213, 31-dihydroxy-GA25). The pumpkin seed 20-oxidase may, there-fore, have different catalytic properties from the equivalentenzyme in those tissues that produce predominantly C19-GAs. The question ofwhether, in the latter case also, a singleenzyme is responsible for each oxidative step at carbon-20 orseparate enzymes catalyze the production of the 10-aldehydeand the loss of carbon-20 should be resolved once GA20-oxidases are cloned from other sources.The N terminus ofthe native 20-oxidase starts at residue 16

of the theoretical sequence, so there may have been somelimited proteolysis during the enzyme purification. The en-zyme is also apparently degraded during two-dimensional gelelectrophoresis with the loss of Mr 15,000 because thisprocedure yielded a major protein of =30,000 Mr (6). Thisresult can now be explained from the derived amino acidsequence, which contains two acid-labile aspartyl-prolinebonds (18). Cleavage of these bonds at low pH duringisoelectric focusing would give three fragments ofMr 28,000,15,000, and 1000.On the basis of their requirement for 2-oxyglutarate, Fe2+

and ascorbate, the soluble GA oxidases, including 2,& and3(3-hydroxylases, as well as the 20-oxidases, are assumed tobelong to a class of plant dioxygenases, many of which use2-oxoglutarate as a cosubstrate (2, 17, 19). The derived aminoacid sequence from the pumpkin 20-oxidase cDNA containsthe regions that are highly conserved in the dioxygenases(Fig. 4), and its membership in this class of enzymes is thusconfirmed. The overall amino acid identity with the otherdioxygenase sequences (<30%o) is typical of that betweenmembers of this class. Higher amino acid-sequence homol-ogy might be expected between the different GA,2-oxo-glutarate dioxygenases, although at present no other se-quences are available for comparison. The derived sequencecontains three conserved histidines, which may serve to bindFe at the enzyme active center (17, 20, 21). The sequencecontains no recognizable N-terminal targeting signals forparticular cell compartments.The potential to examine GA 20-oxidase gene expression in

plant tissues that is now afforded by the availability of thiscDNA is of particular importance. As well as being anessential component of the biosynthesis of C19-GAs, oxida-tion at carbon-20 is thought to be a site of regulation. Forexample, GA 20-oxidase activity is enhanced in leaves of

spinach by exposure to long days, leading to increasedC19-GA production associated with the bolting response (22).Furthermore, the action of GA1 is thought to result indecreased 20-oxidation in a type offeed-back regulation (23).A detailed understanding of these control mechanisms re-quires that the genes encoding GA 20-oxidases are isolatedand characterized. The apparent ease of preparing function-ally active enzyme by heterologous expression, also noted forother plant dioxygenases (13-15), should expedite mechanis-tic and structural studies on this unusual enzyme. In partic-ular, the apparently unique biochemical reaction by whichcarbon-20 is lost will become much more accessible to study.

We thank Dr. A. L. Phillips, J. P. R. Keon, N. E. J. Appleford, Dr.R. Hooley, Dr. A. Bailey, and Dr. J. A. Hargreaves (Long AshtonResearch Station) for valuable advice and assistance, P. Gaskin (LongAshton Research Station) for GC-MS identification ofGA1 and GA2o,S. Barding and B. Schattenberg (Pflanzenphysiologiches Institut,Universitat Gottingen, F.R.G.) for preparing the antibodies, and Dr.L. Hall (Biochemistry Department, University of Bristol, U.K.) forthe DNA sequencing. The work was supportedby the Agricultural andFood Research Council (P.H.), Deutsche Forschungsgemeinschaft(T.L. and J.E.G.), and an Academic Research Collaboration awardfunded by The British Council and Deutsche Akademische Aus-tauschdienst.

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