J. Biochem. 112, 643-651 (1992)
Starch Branching Enzymes from Immature Rice Seeds1 Kouichi Mizuno,' Koji Kimura,* Yuji Arai,* Tsutomu Kawasaki," Hiroaki Shimada," and Tadashi Baba*J 'Institute of Applied Biochemistry, University of Tsukuba, Tsukuba Science City, Ibaraki 305; and "Mitsui Plant Biotechnology Research Institute, Sengen 2-1-6, Tsukuba Science City, Ibaraki 305 Received for publication, June 5, 1992
Four forms of branching enzyme, termed RBE1, RBE2 (a mixture of RBE2A and RBE2B), RBE3, and RBE4, were apparently separated by DEAE-cellulose column chromatography of soluble extract from immature rice seeds, and each of these four forms was further purified by gel-filtration. RBE1, RBE2A, and RBE2B were the predominant forms of the enzyme. The molecular size, amino-terminal amlno acid sequence, and immunoreactivity with anti-maize branching enzyme-I (BE-I) antibody were identical among these three forms, except that the molecular mass of RBE2A was almost 3 kDa higher than those of RBE1 and RBE2B. These results indicate that RBE1, RBE2A, and RBE2B are the same (termed rice BE-I). The cDNA clones coding for rice BE-I have been identified from a rice seed library in Agtll, using the maize BE-I cDNA as a probe. The nucleotide sequence indicates that rice BE-I is initially synthesized as an 820-residue precursor protein, including a putative 64- or 66-residue transit peptide at the amino terminus. The rice mature BE-I contains 756 (or 754) amino acids with a calculated molecular mass of 86,734 (or 86,502) Da, and shares a high degree of sequence identity (86%) with the maize protein. The consensus sequences of the four regions that form the catalytic sites of amylolytic enzymes are conserved in the central region of the rice BE-I sequence. Thus, rice BE-I as well as the maize protein belongs to a family of amylolytic enzymes.
Starch, a storage a-D-glucose polymer in plant, contains two structurally different components, amylose and amylopectin. Amylose is an essentially linear molecule linked by «-l,4-glucosidic bonds, whereas amylopectin aa well as glycogen is highly branched with a-l,6-linked glucosidic linkages. The branches are formed by branching enzyme (a-l,4-D-glucan: a-l,4-D-glucan6-ff-D-glucosyltran8ferase) [EC 2.4.1.18]. Thus, this enzyme plays a key role in amylopectin synthesis. Multiple forms of branching enzyme have been found in various plant tissues (for reviews, see Refs. 1 and 2). There are at least two forms of branching enzyme, BE-I and BE-II, in maize kernels. These two forms are distinguishable from each other in terms of molecular size (82- and 80-kDa for BE-I and BE-II, respectively), amino acid composition, and peptide mapping {3-5). We have also purified maize BE-I completely (6), and identified the cDNA clones from a maize kernel cDNA library (7). The deduced amino acid sequence reveals that plant branching enzyme shares a relatively limited sequence identity with the bacterial enzyme. In rice seeds, two or three forms of branching enzyme have been reported to be present (8). However, the role of each of the enzyme forms in starch synthesis is still unclear. Also, little is known of the structure/function relationship for this enzyme. 1 This study was supported in part by a grant from Iijima Kinen Shokuhin Zaidan. The nucleotide sequence data reported in thin paper will appear in the DDBJ, EMBL, and GenBank Nucleotide Sequence Databases with the following accession number: D11082. 1 To whom all correspondence should be addressed. Abbreviations: bp(s), base pair^s); BE-I, branching enzyme-I; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate.
Vol. 112, No. 5, 1992
643
In order to elucidate further the mechanism of starch synthesis in rice, we have purified and characterized each form of branching enzyme from developing seeds. Moreover, we have characterized the cDNA clones coding for a form of rice branching enzyme corresponding to maize BE-I. MATERIALS AND METHODS Materials—Radioisotopes, [a- 32 P]dCTP (3,000 Ci/ 14 mmol) and a-D-[ C]glucose 1-phosphate (335 mCi/ mmol), were purchased from Bresatec and Amersham, respectively. Nitrocellulose and nylon membranes were purchased from Advantec (Tokyo) and Amersham (Hybond-N), respectively. All other reagents were of the highest purity available. Rice plants, Oryza sativa L. Japonica, were field-grown in 1991 at the Life Science Laboratory of Mitsui Toatsu Chemicals, Mobara. The seeds were obtained at various stages after flowering, immediately frozen in liquid nitrogen, and stored at — 80'C until used. Purification of Branching Enzyme—Frozen rice seeds (200 g, 10-15 days after flowering) were ground in 50 mM Tris/HCl, pH8.5, containing 5mM EDTA and 5mM 2-mercaptoethanol (500 ml) in a mortar and pestle, and homogenized in an electric juicer. The homogenate was filtered through two layers of gauze and centrifuged at 10,000 x g for 20 min. To the supernatant, solid ammonium sulfate was added to give 0-70% saturation, and the mixture was stirred overnight. The pellet was collected by centrifugation at 18,000 Xg, dissolved in a minimum volume of a solution consisting of 50 mM Tris/HCl, pH 7.5, 5 mM EDTA, and 5 mM 2-mercaptoethanol (buffer A), and
644 dialyzed thoroughly against buffer A. The dialyzed solution was applied to a DEAE-cellulose (Whatman DE-52) column previously equilibrated with buffer A. Proteins were eluted with a linear gradient of 0-0.3 M KC1 (2 liters). Fractions (20 ml) were collected at aflowrate of 35 ml/h and assayed for branching enzyme activity. To the pooled fractions containing the enzyme activity, solid ammonium sulfate was added to give 70% saturation. The mixture was stirred for 6 h and centrifuged at 10,000 Xg for lOmin. The precipitate was dissolved in 50 mM Tris/HCl buffer, pH 7.5, containing 5 mM EDTA and 0.2 M KC1 (buffer B), and then applied to a column of Toyopearl HW-55F previously equilibrated with buffer B. Proteins were eluted from the column with buffer B at aflowrate of 10 ml/h. Fractions (3 ml) were collected and assayed for the enzyme activity. All purification procedures were carried out at 0-4*C. Assay for Enzyme Activity—Branching enzyme activity was measured in the direction of stimulation of a-glucan synthesis from aASCAGAiaG»/»CaG«GATCR/MTATCrrGTTGKAffiGCACATASrm6aTIA0GAGTTaGAlG F F A V S S R S G T P E D L K Y L V D K A H S L G L R V L N
1021 270
GATGTTGTCCAT/aaTGDGASTAATAATGTCAaGirGSTCWAATGXmGKGJTGGACWAACACTCATGieiCTTATTrrOT D V V H S H A S N N V T D G L N G Y D V G Q N T H E S Y F H
1111 300
ACAGGAGAT/BG9XT)CCATAAACICTGGGW/CT0GTaBmAACOTGCCAATTffiG«GTCTlAAamOTCTTiaAATTTG T G D R G Y H K L W D S R L F N Y A N W E V L R F L L S N L
1201 330
/«TWTBSATGG(CGMTTCATBTTTGirGKmCGATTTGn'GBGGrrACAraATBCIAW:C»T«CATGGTATCAATA^ R Y M H D E F H F D G F R F D G V T S H L Y H H H G I N K G
1291 360
mACTGGAA«TKA«G«WTTCKTTOG«a-AaGin-GTGGATG»A7TGnT«flGATGClCGaAKCATTOATCOT^ F T G N Y K E Y F S L D T D V D A I V Y H H L A N H L M H K
1381 390
CTCTlGOXGAAGCA/CrArrGTTGCTGfMG?GTTT0GGGCAlGC»GlG(jriCT(SO»GnGATGMQgr&»GlAG9STTTGM: L L P E A T I V A E D V S G M P V L C R P V D E G G V G F D
1471 420
F R L A H A I P D R U I D Y L K N K E D R K U S H S E I V Q
1561 450
T L T N R R Y T E K C I A Y A E S H D Q S I V G D K T I A F
1651 480
L L H D K E M Y T G H S D L Q P A S P T I H R G I A L Q K M
1741 510
ATTCXTTCATTACGATGGDCCTTGGAGGTGCTGXXICTWAfl'TTTATGGXAATGKTTTGHCCATCaGMTCGOTGICTTTOCA I H F I T H A L G G D G Y L N F H G N E F G H P E U I O F P
1831 540
/»eWQBCVCA«TffiAIWGIffAMTa:/«OTC«TBG«;aTGTCGC/CrOTI^Crr(aT«/MeWATB/WG» R E G N N M S Y D K C R R Q U S L V O T D H L R Y K Y H N A
1921 570
TnGVCMGCAA1GAATGCAaCG«GWGMTnK£TlEae7CAinTCAA«CKATrGTTASCGKAlGAKS«AAAGATA« F D Q A H N A L E E E F S F L S S S K Q I V S D H N E K D K
2011 600
GTTATTGKmGMOGTGHGfO'TlGGTTTTTGTTTTCAATTrrCCTOXAVAMACTTKAKGGTTICMAGTCGGAlETGKTm V I V F E R G D L V F V F N F H P N K T Y K G Y K V G C D L
2101 630
CD:G9GVeT«/WGIAGCTOGGCTCTGATGCTTOGTCmeCTGBC(XTQGA/«GTTGECCATSiffGTBGrCXTTC«STa P G K Y R V A L D S D A L V F G G H G R V G H D V D H F T S
2191 660
P E G M P G V P E T N F N N R P N S F K V L S P P R T C V A
2281 690
Y Y R V D E D R E E L R R G G A V A S G K I V T E Y I D V E
2371 720
A
2461 750
T
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TCT BC G»A GAC TQC A M T » S D E D C K *
'
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H
K
G
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E
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S
AGCATWGAT TTCITCATCA GG«DVCIE I H f c m a i TEBATOBG KATCCIB9C TTCCCnBG* Cl PGBIHJIU
2562 756
blltlllAGC /CrnrtATC TMTATCTA 1Q0XIGWC TTTAIBHIA blllUJJLIIA WfiWGWT AffiCKlGtr 9 T C T O X T TWSCCI»G CIGOCMGC
2672
CTAATETA4A MWWGTTT CUtaillCA TDDWATM_AflOBCIGTT CATTDOW CTOWA
2739
Fig. 5. Nucleotide sequence of rice branching enzyme-I and its deduced amino acid sequence. The deduced amino acid sequence is shown below the nucleotide sequence numbered in the 5' to 3' direction. The amino-terminal sequences of RBE1, RBE2A, and RBE2B determined by protein analysis are underlined with a wavy line. The amino acids are numbered from the amino terminus, and the Vol. 112, No. 5, 1992
residues on the amino-terminal side of Thr at residue 1 are indicated by negative numbers. A putative polyadenylation signal is underlined with a broken line. Note that RBE1, RBE2A, and RBE2B possessed two amino-terminal amino acid sequences which started from Thr1 and Val1.
648 R M
K. Mizuno et aL
HLCLTSSSSSAPAPLLP
SLADR-PSPGIAGGGGNVKLSWS
SPRRSWPGKVKTN
-12
IXXVSPSSS-PTPIJTPRRSRSHADRAAPPGIA-
R
R H R
t
LEEFSKCYIJ7anrlVDGATiyREVIAPAA^AQLISEFNNHNSAKHKMEKDKFGIHSIKISH * * ************ * *********** *** ** **** ********* ***** * LESFSK0YIJCT;nrrNEIX?rVYREHAPAAQHELI(3)nn^^ VNGKPAIPHHSKVKTRTRHGCKAHVDRIPAKIRyATFnASKrGAPYIXWHHDPPACBRTVrK * *************** * ** ******* ***** ****************** *** **
55 110 117 172
M VKGKPAIPHNSKVKnirLH-AAPRiyEAHV(WSGEKPAVSTYREFADKVLPRIRANNYNTVQLMAVMEHSYyASF
240
Fig. 6. Comparison of deduced amino acid sequences between rice (R) and maize (M) branching enzyme-I precursors. Amino acids are represented by the standard one-letter codes. Identical residues are indicated by asterisks. Dashes represent gaps introduced to maximize the alignment similarity. Arrows indicate the amino-terminal amino acids of the rice and mni7j> mature proteins. The boxed sequences of the rice and maize proteins share a high degree of identity with the sequences of four active-site regions of amylolytic enzymes (regions I, II, HI, and IV, see Refs. 26 and 27). The maize sequence is that reported by Baba et al (7).
I, M JWHS^SNNVTDGLNGYDVGQSTQE R CTHVTMTAVSSRSGTPEDLKXLVDKAHSLGLRVIJ' CTHVTNFFAVSSRSGTPKO^YLVDKAHSI/ILRVUfJWHS^ISNNVT^ R SYTHT(a»GyHKLHDSRUNYANWEVLRrLLSNLRyi«DE M SYFHASOAI,VrG(aGRVCTDVDHrrS PEGMPGVP
668
M
GDLVrVFNTHPKKTYEGYKVGCDLPGKYRVALDSDALVFtXfflGRWaDVDHFTSPEGVPGVP
674
R
ETNTlJNRPNSrKVI£PPRTCVAYTRVDEDREELRRGGAVA-SGK-IVTEYIDVEATSGETIS **************************** ** * * ** * *** * * * ETNFNNRPKSTKVI£PPRTCVAyyRVDE-A«a«3«RLHAKAFrcKTSPAESICWKA-SRA-SS GGMraSEKDDCGKKOaOVFRSSDEDCK * * **** ** ** * * KEDK~EATAGGKR3WKrARQPSDQDTK
M R M
clones, termed MB9 and MB10, coding for maize BE-I (7). The cDNA insert of MB9 was 32P-labeled and used to screen approximately 7.5 X106 recombinant phages from a rice seed cDNA library in Agtll. Twenty positive clones were obtained after two more rounds of screening. Restriction mapping and Southern blot analysis of these cDNA inserts showed that they were all related. Therefore, we selected at random three clones, termed RB12, RB13, and RB15, for further characterization. The restriction map and sequencing strategy used are given in Fig. 4. Of these three clones selected, RB13 contained the longest cDNA insert with a size of approximately 2.7 kbp. The nucleotide sequence of the overlapping cDNA inserts contains a 2,460-nucleotide open reading frame, which is flanked by 19-nucleotide 5'-untranslated and 257-nucleotide 3'-untranslated regions (Fig. 5). The ATG start codon is assigned on the basis of its similarity to the eukaryotic consensus sequence, as described by Kozak (18). A putative polyadenylatdon signal, AATAAA, is located at nucleotides 2,683-2,688 and 2,708-2,713. The 17-residue sequence at residues 1-17 matches the amino-terminal sequences of RBE1, RBE2A, and RBE2B determined experimentally (Fig. 5). This result confirms that the isolated cDNA clones encode RBE1 as well as RBE2A and RBE2B (to avoid complexity, the term, BE-I, is used for RBEl, RBE2A, and RBE2B hereafter). The cDNA-derived sequence indicates that rice BE-I is initially synthesized as an 820-residue polypeptide, and that the
728 733 756 759
mature protein contains 756 (or 754) amino acids, including 9 cysteine residues, with a calculated molecular mass of 86,734 (or 86,502) Da (Fig. 5). This value is consistent with that determined for RBE2A by SDS-PAGE (Fig. 3). The rice BE-I precursor as well as the maize protein possesses no typical signal peptide sequence (Figs. 5 and 6). It is most likely that the branching enzyme possesses a leader sequence necessary for transport into the amyloplast. The 64-residue sequence at residues —64 to —1 preceding the amino terminus of the mature rice BE-I is rich in hydroxylic (23% as Ser and Thr) and basic (14% as Arg and Lys) amino acids, and contains an acidic amino acid (Asp) only at residue —44 (Figs. 5 and 6). Thus, the amino-terminal leader peptide of the rice BE-I precursor is similar in amino acid composition to the transit peptide of potato phosphorylase precursor, which is nuclear-encoded and transported into the amyloplast (19, 20). The putative transit peptide of the rice BE-I precursor shares a significant degree of sequence identity (53%) with that of the maize protein (Fig. 6), but not with those of ADP-glucose pyrophosphorylase (21) and waxy protein (Ref. 22, data not shown). Sequence alignment of mature rice BE-I to the maize protein shows 86% identity (Fig. 6). Interestingly, the carboxy-terminal region is poorly conserved. Rice BE-I contains nine cysteine residues, five of which are conserved between the rice and maize proteins. Since plant branching enzyme requires free sulfhydryl groups for activity (23), J. Biochem.
649
Branching Enzymes from Rice Seeds R E
TMVTWEEVDH-LPtYDLD PKLE EFKDHFNYRIK— MSDRIDRDVINAIJJ«aFADPFSVIX34Hin1*Ai3L**RAL**I»T-*VWVIEPKTGRKLA***CLDSRG*FSGVIP*R*Nr
33 79
R E
-RY LDQKCLI EKHEGGLEE FSKGYLKFGINTVDGATIYR—EWAPAAQEAQLIGEFNNW F**QLAWWHG*QN**DDPYRFGPLIQ*MDAWL*S*GTHLRPyET*GAHAD*M**V*GT*FSV***N*RRVSW*Q**Y*
89 159
R E
NGAKHKMEKDK-FGIWIIU-SHVNGKPMPHNSKvTFRFRHGGGAWVTlRIPAWIRYATFDAS-!^^ D*RR*P*RLR*ES***ELF*PGAH**QLYKYEMIDANOIL*—LKSDPYAFE*QH*PE*~**LIC*L*EKV*
QTE
166 232
R E P
ERYVFKHPRPPKPDAP-RIYEAHVG MSGEEPEVSTYREFADNVLPRIRANNYNTVQLMAIMEHSYYASFGYHVTNFF ** KKANQF***IS***V*L*SKRRHTDNNFWLS***L**QLV*YAKNMGFTHLE*LP*N**PFDG*W**QP*GLY **R*HS******D*****K******************G**********
242 306 325
R E P
AVSSRSGTPEDLKYLVDKAHSU3LRVT* D W H S H KSNNVTDGLNGYDVGQNTHESYFHTGDR-GYHKLWDSRLFNYANWE *PTR*F**RD*FR*FI*A**AA**N*II •W*PG* —FPTD*FALAEFD*T* L*E*SDP*E***QD*NTUY**GRR* *Y*N*******X********Q**V ****** fc**********p*i**QSQ*****j^*g*»*****************
321 381 404
R E P
VLRFLLSNLRYHMDEFMFT 3FRFDGVTS HLYHHBGINKGFTGNYJ 3YFS LXmJVTlAIVYMMLANHUffilCLLPEATIVAED *SN**VG*AL**IER*GI* fcL*V*A*A* *I*RDY3RKE*-EWIPN *FGG -RENLE**EFLRNT*RILGEQVSG*VTM**E * * * * * * * * ! * * ***v*****M******N **** 5A*****v**L****N*I**IF*D**VI*** ********* *
401 459 484
R E P
VSGAO^TLOlPVDEGGVGFDFRLAMAIPDRWIDYIJaJKEDRKVISMSEIVQTLTNRRYTEKCIA YAESHC 3SIVGDKTIAFL STDF*GVS**Q*M**L**WYKWNLGW4HDYL**M*LDPVYRQYHHDKLTFGILYN***-NFVLPL***EWH*K*S* *****Q*Q***S***j***Y********K********N*ED*
481 535 527
R E
umKEMYTGMSDLQPASPTIfn^IAIjQKMIHFITMALGGDGYLNFMGNEFGHP-EWI-D FPREG-NNWSYDKCRR LDRMP*DAWQKF* *LRAYYGH*KA*PGKK *L******AQGR**NH*ASLDWHLL**GD**HHGVQ*L
553 602
R E
QHSLVDT-DHLRYKYMNAFIX^MHALEEEFSFLSSSKQIVSDMHEKDKVIVFERGDLVFVFNFHPNKTYKGYKVGCDLPG VRD*NL*YR*HKAMHELD**PYG7EWLWDDKER*VLIF*RRDK*GNEI** AS**T*-VPRHD*RF*INQ**
632 673
R E
KYRVALDSDALVFGGHGRVGHDVDHFTSPEGMP
Starch Branching Enzymes from Immature Rice Seeds1 Kouichi Mizuno,' Koji Kimura,* Yuji Arai,* Tsutomu Kawasaki," Hiroaki Shimada," and Tadashi Baba*J 'Institute of Applied Biochemistry, University of Tsukuba, Tsukuba Science City, Ibaraki 305; and "Mitsui Plant Biotechnology Research Institute, Sengen 2-1-6, Tsukuba Science City, Ibaraki 305 Received for publication, June 5, 1992
Four forms of branching enzyme, termed RBE1, RBE2 (a mixture of RBE2A and RBE2B), RBE3, and RBE4, were apparently separated by DEAE-cellulose column chromatography of soluble extract from immature rice seeds, and each of these four forms was further purified by gel-filtration. RBE1, RBE2A, and RBE2B were the predominant forms of the enzyme. The molecular size, amino-terminal amlno acid sequence, and immunoreactivity with anti-maize branching enzyme-I (BE-I) antibody were identical among these three forms, except that the molecular mass of RBE2A was almost 3 kDa higher than those of RBE1 and RBE2B. These results indicate that RBE1, RBE2A, and RBE2B are the same (termed rice BE-I). The cDNA clones coding for rice BE-I have been identified from a rice seed library in Agtll, using the maize BE-I cDNA as a probe. The nucleotide sequence indicates that rice BE-I is initially synthesized as an 820-residue precursor protein, including a putative 64- or 66-residue transit peptide at the amino terminus. The rice mature BE-I contains 756 (or 754) amino acids with a calculated molecular mass of 86,734 (or 86,502) Da, and shares a high degree of sequence identity (86%) with the maize protein. The consensus sequences of the four regions that form the catalytic sites of amylolytic enzymes are conserved in the central region of the rice BE-I sequence. Thus, rice BE-I as well as the maize protein belongs to a family of amylolytic enzymes.
Starch, a storage a-D-glucose polymer in plant, contains two structurally different components, amylose and amylopectin. Amylose is an essentially linear molecule linked by «-l,4-glucosidic bonds, whereas amylopectin aa well as glycogen is highly branched with a-l,6-linked glucosidic linkages. The branches are formed by branching enzyme (a-l,4-D-glucan: a-l,4-D-glucan6-ff-D-glucosyltran8ferase) [EC 2.4.1.18]. Thus, this enzyme plays a key role in amylopectin synthesis. Multiple forms of branching enzyme have been found in various plant tissues (for reviews, see Refs. 1 and 2). There are at least two forms of branching enzyme, BE-I and BE-II, in maize kernels. These two forms are distinguishable from each other in terms of molecular size (82- and 80-kDa for BE-I and BE-II, respectively), amino acid composition, and peptide mapping {3-5). We have also purified maize BE-I completely (6), and identified the cDNA clones from a maize kernel cDNA library (7). The deduced amino acid sequence reveals that plant branching enzyme shares a relatively limited sequence identity with the bacterial enzyme. In rice seeds, two or three forms of branching enzyme have been reported to be present (8). However, the role of each of the enzyme forms in starch synthesis is still unclear. Also, little is known of the structure/function relationship for this enzyme. 1 This study was supported in part by a grant from Iijima Kinen Shokuhin Zaidan. The nucleotide sequence data reported in thin paper will appear in the DDBJ, EMBL, and GenBank Nucleotide Sequence Databases with the following accession number: D11082. 1 To whom all correspondence should be addressed. Abbreviations: bp(s), base pair^s); BE-I, branching enzyme-I; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate.
Vol. 112, No. 5, 1992
643
In order to elucidate further the mechanism of starch synthesis in rice, we have purified and characterized each form of branching enzyme from developing seeds. Moreover, we have characterized the cDNA clones coding for a form of rice branching enzyme corresponding to maize BE-I. MATERIALS AND METHODS Materials—Radioisotopes, [a- 32 P]dCTP (3,000 Ci/ 14 mmol) and a-D-[ C]glucose 1-phosphate (335 mCi/ mmol), were purchased from Bresatec and Amersham, respectively. Nitrocellulose and nylon membranes were purchased from Advantec (Tokyo) and Amersham (Hybond-N), respectively. All other reagents were of the highest purity available. Rice plants, Oryza sativa L. Japonica, were field-grown in 1991 at the Life Science Laboratory of Mitsui Toatsu Chemicals, Mobara. The seeds were obtained at various stages after flowering, immediately frozen in liquid nitrogen, and stored at — 80'C until used. Purification of Branching Enzyme—Frozen rice seeds (200 g, 10-15 days after flowering) were ground in 50 mM Tris/HCl, pH8.5, containing 5mM EDTA and 5mM 2-mercaptoethanol (500 ml) in a mortar and pestle, and homogenized in an electric juicer. The homogenate was filtered through two layers of gauze and centrifuged at 10,000 x g for 20 min. To the supernatant, solid ammonium sulfate was added to give 0-70% saturation, and the mixture was stirred overnight. The pellet was collected by centrifugation at 18,000 Xg, dissolved in a minimum volume of a solution consisting of 50 mM Tris/HCl, pH 7.5, 5 mM EDTA, and 5 mM 2-mercaptoethanol (buffer A), and
644 dialyzed thoroughly against buffer A. The dialyzed solution was applied to a DEAE-cellulose (Whatman DE-52) column previously equilibrated with buffer A. Proteins were eluted with a linear gradient of 0-0.3 M KC1 (2 liters). Fractions (20 ml) were collected at aflowrate of 35 ml/h and assayed for branching enzyme activity. To the pooled fractions containing the enzyme activity, solid ammonium sulfate was added to give 70% saturation. The mixture was stirred for 6 h and centrifuged at 10,000 Xg for lOmin. The precipitate was dissolved in 50 mM Tris/HCl buffer, pH 7.5, containing 5 mM EDTA and 0.2 M KC1 (buffer B), and then applied to a column of Toyopearl HW-55F previously equilibrated with buffer B. Proteins were eluted from the column with buffer B at aflowrate of 10 ml/h. Fractions (3 ml) were collected and assayed for the enzyme activity. All purification procedures were carried out at 0-4*C. Assay for Enzyme Activity—Branching enzyme activity was measured in the direction of stimulation of a-glucan synthesis from aASCAGAiaG»/»CaG«GATCR/MTATCrrGTTGKAffiGCACATASrm6aTIA0GAGTTaGAlG F F A V S S R S G T P E D L K Y L V D K A H S L G L R V L N
1021 270
GATGTTGTCCAT/aaTGDGASTAATAATGTCAaGirGSTCWAATGXmGKGJTGGACWAACACTCATGieiCTTATTrrOT D V V H S H A S N N V T D G L N G Y D V G Q N T H E S Y F H
1111 300
ACAGGAGAT/BG9XT)CCATAAACICTGGGW/CT0GTaBmAACOTGCCAATTffiG«GTCTlAAamOTCTTiaAATTTG T G D R G Y H K L W D S R L F N Y A N W E V L R F L L S N L
1201 330
/«TWTBSATGG(CGMTTCATBTTTGirGKmCGATTTGn'GBGGrrACAraATBCIAW:C»T«CATGGTATCAATA^ R Y M H D E F H F D G F R F D G V T S H L Y H H H G I N K G
1291 360
mACTGGAA«TKA«G«WTTCKTTOG«a-AaGin-GTGGATG»A7TGnT«flGATGClCGaAKCATTOATCOT^ F T G N Y K E Y F S L D T D V D A I V Y H H L A N H L M H K
1381 390
CTCTlGOXGAAGCA/CrArrGTTGCTGfMG?GTTT0GGGCAlGC»GlG(jriCT(SO»GnGATGMQgr&»GlAG9STTTGM: L L P E A T I V A E D V S G M P V L C R P V D E G G V G F D
1471 420
F R L A H A I P D R U I D Y L K N K E D R K U S H S E I V Q
1561 450
T L T N R R Y T E K C I A Y A E S H D Q S I V G D K T I A F
1651 480
L L H D K E M Y T G H S D L Q P A S P T I H R G I A L Q K M
1741 510
ATTCXTTCATTACGATGGDCCTTGGAGGTGCTGXXICTWAfl'TTTATGGXAATGKTTTGHCCATCaGMTCGOTGICTTTOCA I H F I T H A L G G D G Y L N F H G N E F G H P E U I O F P
1831 540
/»eWQBCVCA«TffiAIWGIffAMTa:/«OTC«TBG«;aTGTCGC/CrOTI^Crr(aT«/MeWATB/WG» R E G N N M S Y D K C R R Q U S L V O T D H L R Y K Y H N A
1921 570
TnGVCMGCAA1GAATGCAaCG«GWGMTnK£TlEae7CAinTCAA«CKATrGTTASCGKAlGAKS«AAAGATA« F D Q A H N A L E E E F S F L S S S K Q I V S D H N E K D K
2011 600
GTTATTGKmGMOGTGHGfO'TlGGTTTTTGTTTTCAATTrrCCTOXAVAMACTTKAKGGTTICMAGTCGGAlETGKTm V I V F E R G D L V F V F N F H P N K T Y K G Y K V G C D L
2101 630
CD:G9GVeT«/WGIAGCTOGGCTCTGATGCTTOGTCmeCTGBC(XTQGA/«GTTGECCATSiffGTBGrCXTTC«STa P G K Y R V A L D S D A L V F G G H G R V G H D V D H F T S
2191 660
P E G M P G V P E T N F N N R P N S F K V L S P P R T C V A
2281 690
Y Y R V D E D R E E L R R G G A V A S G K I V T E Y I D V E
2371 720
A
2461 750
T
S
G
E
T
I
S
G
TCT BC G»A GAC TQC A M T » S D E D C K *
'
G
H
K
G
S
E
K
D
D
C
G
K
K
G
H
K
F
V
F
R
S
AGCATWGAT TTCITCATCA GG«DVCIE I H f c m a i TEBATOBG KATCCIB9C TTCCCnBG* Cl PGBIHJIU
2562 756
blltlllAGC /CrnrtATC TMTATCTA 1Q0XIGWC TTTAIBHIA blllUJJLIIA WfiWGWT AffiCKlGtr 9 T C T O X T TWSCCI»G CIGOCMGC
2672
CTAATETA4A MWWGTTT CUtaillCA TDDWATM_AflOBCIGTT CATTDOW CTOWA
2739
Fig. 5. Nucleotide sequence of rice branching enzyme-I and its deduced amino acid sequence. The deduced amino acid sequence is shown below the nucleotide sequence numbered in the 5' to 3' direction. The amino-terminal sequences of RBE1, RBE2A, and RBE2B determined by protein analysis are underlined with a wavy line. The amino acids are numbered from the amino terminus, and the Vol. 112, No. 5, 1992
residues on the amino-terminal side of Thr at residue 1 are indicated by negative numbers. A putative polyadenylation signal is underlined with a broken line. Note that RBE1, RBE2A, and RBE2B possessed two amino-terminal amino acid sequences which started from Thr1 and Val1.
648 R M
K. Mizuno et aL
HLCLTSSSSSAPAPLLP
SLADR-PSPGIAGGGGNVKLSWS
SPRRSWPGKVKTN
-12
IXXVSPSSS-PTPIJTPRRSRSHADRAAPPGIA-
R
R H R
t
LEEFSKCYIJ7anrlVDGATiyREVIAPAA^AQLISEFNNHNSAKHKMEKDKFGIHSIKISH * * ************ * *********** *** ** **** ********* ***** * LESFSK0YIJCT;nrrNEIX?rVYREHAPAAQHELI(3)nn^^ VNGKPAIPHHSKVKTRTRHGCKAHVDRIPAKIRyATFnASKrGAPYIXWHHDPPACBRTVrK * *************** * ** ******* ***** ****************** *** **
55 110 117 172
M VKGKPAIPHNSKVKnirLH-AAPRiyEAHV(WSGEKPAVSTYREFADKVLPRIRANNYNTVQLMAVMEHSYyASF
240
Fig. 6. Comparison of deduced amino acid sequences between rice (R) and maize (M) branching enzyme-I precursors. Amino acids are represented by the standard one-letter codes. Identical residues are indicated by asterisks. Dashes represent gaps introduced to maximize the alignment similarity. Arrows indicate the amino-terminal amino acids of the rice and mni7j> mature proteins. The boxed sequences of the rice and maize proteins share a high degree of identity with the sequences of four active-site regions of amylolytic enzymes (regions I, II, HI, and IV, see Refs. 26 and 27). The maize sequence is that reported by Baba et al (7).
I, M JWHS^SNNVTDGLNGYDVGQSTQE R CTHVTMTAVSSRSGTPEDLKXLVDKAHSLGLRVIJ' CTHVTNFFAVSSRSGTPKO^YLVDKAHSI/ILRVUfJWHS^ISNNVT^ R SYTHT(a»GyHKLHDSRUNYANWEVLRrLLSNLRyi«DE M SYFHASOAI,VrG(aGRVCTDVDHrrS PEGMPGVP
668
M
GDLVrVFNTHPKKTYEGYKVGCDLPGKYRVALDSDALVFtXfflGRWaDVDHFTSPEGVPGVP
674
R
ETNTlJNRPNSrKVI£PPRTCVAYTRVDEDREELRRGGAVA-SGK-IVTEYIDVEATSGETIS **************************** ** * * ** * *** * * * ETNFNNRPKSTKVI£PPRTCVAyyRVDE-A«a«3«RLHAKAFrcKTSPAESICWKA-SRA-SS GGMraSEKDDCGKKOaOVFRSSDEDCK * * **** ** ** * * KEDK~EATAGGKR3WKrARQPSDQDTK
M R M
clones, termed MB9 and MB10, coding for maize BE-I (7). The cDNA insert of MB9 was 32P-labeled and used to screen approximately 7.5 X106 recombinant phages from a rice seed cDNA library in Agtll. Twenty positive clones were obtained after two more rounds of screening. Restriction mapping and Southern blot analysis of these cDNA inserts showed that they were all related. Therefore, we selected at random three clones, termed RB12, RB13, and RB15, for further characterization. The restriction map and sequencing strategy used are given in Fig. 4. Of these three clones selected, RB13 contained the longest cDNA insert with a size of approximately 2.7 kbp. The nucleotide sequence of the overlapping cDNA inserts contains a 2,460-nucleotide open reading frame, which is flanked by 19-nucleotide 5'-untranslated and 257-nucleotide 3'-untranslated regions (Fig. 5). The ATG start codon is assigned on the basis of its similarity to the eukaryotic consensus sequence, as described by Kozak (18). A putative polyadenylatdon signal, AATAAA, is located at nucleotides 2,683-2,688 and 2,708-2,713. The 17-residue sequence at residues 1-17 matches the amino-terminal sequences of RBE1, RBE2A, and RBE2B determined experimentally (Fig. 5). This result confirms that the isolated cDNA clones encode RBE1 as well as RBE2A and RBE2B (to avoid complexity, the term, BE-I, is used for RBEl, RBE2A, and RBE2B hereafter). The cDNA-derived sequence indicates that rice BE-I is initially synthesized as an 820-residue polypeptide, and that the
728 733 756 759
mature protein contains 756 (or 754) amino acids, including 9 cysteine residues, with a calculated molecular mass of 86,734 (or 86,502) Da (Fig. 5). This value is consistent with that determined for RBE2A by SDS-PAGE (Fig. 3). The rice BE-I precursor as well as the maize protein possesses no typical signal peptide sequence (Figs. 5 and 6). It is most likely that the branching enzyme possesses a leader sequence necessary for transport into the amyloplast. The 64-residue sequence at residues —64 to —1 preceding the amino terminus of the mature rice BE-I is rich in hydroxylic (23% as Ser and Thr) and basic (14% as Arg and Lys) amino acids, and contains an acidic amino acid (Asp) only at residue —44 (Figs. 5 and 6). Thus, the amino-terminal leader peptide of the rice BE-I precursor is similar in amino acid composition to the transit peptide of potato phosphorylase precursor, which is nuclear-encoded and transported into the amyloplast (19, 20). The putative transit peptide of the rice BE-I precursor shares a significant degree of sequence identity (53%) with that of the maize protein (Fig. 6), but not with those of ADP-glucose pyrophosphorylase (21) and waxy protein (Ref. 22, data not shown). Sequence alignment of mature rice BE-I to the maize protein shows 86% identity (Fig. 6). Interestingly, the carboxy-terminal region is poorly conserved. Rice BE-I contains nine cysteine residues, five of which are conserved between the rice and maize proteins. Since plant branching enzyme requires free sulfhydryl groups for activity (23), J. Biochem.
649
Branching Enzymes from Rice Seeds R E
TMVTWEEVDH-LPtYDLD PKLE EFKDHFNYRIK— MSDRIDRDVINAIJJ«aFADPFSVIX34Hin1*Ai3L**RAL**I»T-*VWVIEPKTGRKLA***CLDSRG*FSGVIP*R*Nr
33 79
R E
-RY LDQKCLI EKHEGGLEE FSKGYLKFGINTVDGATIYR—EWAPAAQEAQLIGEFNNW F**QLAWWHG*QN**DDPYRFGPLIQ*MDAWL*S*GTHLRPyET*GAHAD*M**V*GT*FSV***N*RRVSW*Q**Y*
89 159
R E
NGAKHKMEKDK-FGIWIIU-SHVNGKPMPHNSKvTFRFRHGGGAWVTlRIPAWIRYATFDAS-!^^ D*RR*P*RLR*ES***ELF*PGAH**QLYKYEMIDANOIL*—LKSDPYAFE*QH*PE*~**LIC*L*EKV*
QTE
166 232
R E P
ERYVFKHPRPPKPDAP-RIYEAHVG MSGEEPEVSTYREFADNVLPRIRANNYNTVQLMAIMEHSYYASFGYHVTNFF ** KKANQF***IS***V*L*SKRRHTDNNFWLS***L**QLV*YAKNMGFTHLE*LP*N**PFDG*W**QP*GLY **R*HS******D*****K******************G**********
242 306 325
R E P
AVSSRSGTPEDLKYLVDKAHSU3LRVT* D W H S H KSNNVTDGLNGYDVGQNTHESYFHTGDR-GYHKLWDSRLFNYANWE *PTR*F**RD*FR*FI*A**AA**N*II •W*PG* —FPTD*FALAEFD*T* L*E*SDP*E***QD*NTUY**GRR* *Y*N*******X********Q**V ****** fc**********p*i**QSQ*****j^*g*»*****************
321 381 404
R E P
VLRFLLSNLRYHMDEFMFT 3FRFDGVTS HLYHHBGINKGFTGNYJ 3YFS LXmJVTlAIVYMMLANHUffilCLLPEATIVAED *SN**VG*AL**IER*GI* fcL*V*A*A* *I*RDY3RKE*-EWIPN *FGG -RENLE**EFLRNT*RILGEQVSG*VTM**E * * * * * * * * ! * * ***v*****M******N **** 5A*****v**L****N*I**IF*D**VI*** ********* *
401 459 484
R E P
VSGAO^TLOlPVDEGGVGFDFRLAMAIPDRWIDYIJaJKEDRKVISMSEIVQTLTNRRYTEKCIA YAESHC 3SIVGDKTIAFL STDF*GVS**Q*M**L**WYKWNLGW4HDYL**M*LDPVYRQYHHDKLTFGILYN***-NFVLPL***EWH*K*S* *****Q*Q***S***j***Y********K********N*ED*
481 535 527
R E
umKEMYTGMSDLQPASPTIfn^IAIjQKMIHFITMALGGDGYLNFMGNEFGHP-EWI-D FPREG-NNWSYDKCRR LDRMP*DAWQKF* *LRAYYGH*KA*PGKK *L******AQGR**NH*ASLDWHLL**GD**HHGVQ*L
553 602
R E
QHSLVDT-DHLRYKYMNAFIX^MHALEEEFSFLSSSKQIVSDMHEKDKVIVFERGDLVFVFNFHPNKTYKGYKVGCDLPG VRD*NL*YR*HKAMHELD**PYG7EWLWDDKER*VLIF*RRDK*GNEI** AS**T*-VPRHD*RF*INQ**
632 673
R E
KYRVALDSDALVFGGHGRVGHDVDHFTSPEGMP
