Plant Molecular Biology 17: 1089-1093, 1991. © 1991 Kluwer Academic Publishers. Printed in Belgium.
1089
Update section
Short communication
Comparison of the primary sequences of two potato tuber ADP-glucose pyrophosphorylase subunits Paul A. Nakata, 1 Thomas W. Greene, 2 Joseph M. Anderson, 3 Brian J. Smith-White,4 Thomas W. Okita 3. and Jack Preiss 4 1Program in Biochemistry and Biophysics, 2program in Plant Physiology, and 3Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA (*author for correspondence); 4Department of Biochemistry, Michigan State University, East Lansing, MI 48824, USA Received 3 July 1991; accepted 9 July 1991
Key words: ADP-glucose pyrophosphorylase, potato, starch biosynthesis
Abstract Near-full-length cDNA clones to the small and large subunit of the heterotetrameric potato tuber ADP-glucose pyrophosphorylase have been isolated and characterized. The missing amino terminal sequence of the small subunit has also been elucidated from its corresponding genomic clone. Primary sequence comparisons revealed that each potato subunit had less identity to each other than to their homologous subunit from other plants. It also appeared that the smaller subunit is more conserved among the different plants and the larger subunit more divergent. Amino acid comparisons of both potato tuber sequences to the Escherichia coli ADP-glucose pyrophosphorylase sequence revealed conserved regions important for both catalytic and allosteric function of the bacterial enzyme.
ADP-glucose pyrophosphorylase plays a pivotal role in the biosynthesis of ~l,4-glucans in both bacteria [19] and plants [20, 21, 25]. This tetrameric enzyme mediates the synthesis of the activated glucosyl donor, ADP-glucose, from glucose- 1-P and ATP. The catalytic activity of both the bacterial and plant enzymes are allosterically regulated by small effector molecules which oscillate during normal carbon metabolism in these organisms [18]. In Escherichia coli, ADP-glucose
pyrophosphorylase is activated by fructose-l,6P2 and inhibited by AMP and ADP, whereas the plant enzyme is stimulated by 3-PGA and inhibited by Pi [ 18]. Fluctuation in the 3-PGA/Pi ratio, in response to photosynthesis, has been shown to regulate the transient production of starch in leaf tissue [6]. Whether this allosteric regulation operates in non-photosynthetic tissue is not known. To gain insight into the structure/function relationship of the plant enzyme we have elucidated
The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases.
1090
the heterotetrameric structure of the potato tuber enzyme [15] and have isolated near-full-length c D N A clones to the small subunit as previously reported [2]. Our findings have been corroborated by other groups [5, 14]. Recently we have isolated and characterized a near-full-length c D N A clone to the large subunit of the tuber enzyme and have derived the missing N-terminal coding sequence of the small subunit from its corresponding genomic clone. A comparison of the small and large potato tuber subunits revealed a 52~o amino acid identity overall which is somewhat higher than the 35~o identity observed between the spinach leaf subunits [22]. The potato small subunit showed 8 4 ~ identity to the rice endosperm small subunit [ 1 ] and 93 ~o identity to the spinach leaf small subunit [22] at the primary level. The potato large subunit exhibited 58 ~o and 61~o sequence identity to AGA.7 [16] and shrunken-2 [4] clones, which encode the wheat i
Consensus Wheat Maize Potato
K..... R. . . . $ . . . . . Q. . . . . . R ..NG .......... TT . . . . . T ...... R. R ASPPSESRAP LIb~PORSATR OHOAROGPRR MCNGGRGPPY WTAGVTSAPA RQTPLFSGRP
NSGGKCDFDL A A T F F C S W Y L W G G D M Q F A LALDTNSGPH QIRSCEGDGI DRLEKLSIGG ~KQEKALRNR CFGGRVAATQ QCILTSDCAF ETLHSQTOSS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . NKIKPGVA YSVITTENDT QTVFVDMPRL
.... AOP..V SGGLSDPNEV RKNYADNARV ERRRANPKDV
E.AG..WFQG EAAG..WFRG EPAG.,WFQG E.AGKKWFQG
.
CFNSGINKIF CFNSGINKIF CFNSGINKIF CINSAINKIF
VMTQFNSASL VMTQFNSASL VMSQFNSTSL VLTQYNSAPL
NRHIHRTYLG NRHIHRTYLG NRHIHRTYLE NRHIARTYFG
GGINF.DGSV GGINFTDGSV GGINFADGSV NGVSFGDGFV
VQKHVDDNAD VQKHVDDNAD VQKHVEDDAD VQNHIDRNAD
ITLSCAPV.E ITLSCAPVGE ITISCAPVDE ITLSCAPAED
SRAS..GLVK SRASEYGLVK SRASKNGLVK SRASDFGLVK
I D S . G R W Q F ..EKPKG.DL F D S S G R W Q F SAEQPKGDDL IDHTGRVLOP P.EKPKGADL I D S R G R W Q F A.EKPKGFDL
AAVILGGGTG AAVILGGGTG SAIILGGGTG AAVILGGGEG
TQLFPLTSTR TQLFPLTSTR SOLFPLTSTR TKLFPLTSRT
ATPAVPVGGC ATPAVPIGGC ATPAVPVGGC ATPAVPVGGC
YRLIDIPMSN YRLIDIPMSN YRLIDIPMSN YRLIDIPMSN
TADA.RKFIW TAOAWRKIIW TAOSIRKFIW TADAVRKFIW
VLEDYY..KS VLEDYYKNKS VLEDYYSHKS VFEDAKNKNI
I..IVILSGD IEHILILSGD IDNIVILSGD ENIW.LSGD
QLYRMDYMEL QLYRMDYMEL QLYRMNYMEL HLYRMDYMEL
.AM.VDT.FL EAMKVDTSFL NSMRVETNFL KAMQVDTTLV
Consensus Wheat Maize Potato
PKTPFFTSPR PKTPFFTSFR PKTPFFTAPR PKTPFYTSPR
..AIDDA.K¥ NFAIDDPAKY SYAIDDAQKY GLSPQOAKK$
PYIASMGVYV PYIASMGVyV PYLASMGIYV PYIASMGVyV
FK.DVLL.LL FKRDVLLNLL FKKDALLDLL FRTDVLLKLL
KS.Y..LHDF KSRYAELHDF KSKYTQLHDF KWSYPTSNDF
.LPPTK.DKC YLPPTKSDKC CLPPTQLDKC FLPPTKIDNC
KIK.AIISHG RIKEAIILHG KMRYAFISDG KIKDAIISHG
CFLREC. IEH CFLRECKIEH CLLRECNIEH CFLRDCSVEH
S..G..SRL. TAF...SRLN SVIGVCSRVS $1VGERSRLD
,
• ,i
t
Ittl
***
,
•
t,
,
K.GVQEADRP KEGVQEADRP SKGIQEADHP KDGVQEADRP
EEGYYIRSGI %e41LKNATI. DGTVI EEGYYIRSGI W I Q K N A T I K D G T W EEGYYIRSGI W I L K N A T I N ECLVI EEGFYIRSGI IIILEKATIR DGTVI
***t,t
,
EVLAATQMPG EVLAATQMPG QVLAATQMPE EVLAATQTPG 300
400 GSEILPRA.. GSEILPRALH GSEILPRAVL GSEIIPAAID
DHNVQAYIFT DHNVQAYVFT DHSVQACIFT DYNVQAYIFK
DYWEDIGT~K DYWEDIGTIR GYWEDVGTIK DYWEDIGTIK
SFFDANLALT SFFDANRALC SFFDANLALT SFYNASLALT
EQPPKF.FYD EQPPKFEFYD EQPSKFDFYD QE?PEFQFYD
SG.ELKD..M SGSELKNAMM SGCELKDSVM CGVELKDTFM
MGAD.YETE. MGADSYETED MGADIYETEE MGADYYQTES
E.S.LL.EGK EMSRLMSEGK EASKLLLAGK EIASLLAEGK
VPIGIGENTK VPIGVGENTK VPIGIGRNTK VPIGIGENTK
IRNCIIDMNA ISNCIIDMNA IRNCIIDMNA IRKCIIDKNA
500
450
545
501 RIGKNVVI.N RIGRDWISN RIGKNWITN KZGKNVSIIN
.it**
350
401
Consensus Wheat Maize Potato
t*
250
301
Consensus Wheat Maize Potato
200
150 **********
201 Consensus Wheat Maize Potato
I00
50
............................................. .......................................
101 Consensus Wheat Maize Potato
and maize endosperm large subunits, respectively (Fig. 1). There appears to be less identity between the two subunits from the same species than between similar subunits from the different plants. The identity studies also suggest that the smaller subunits are more conserved among the different plants and the larger subunits more divergent. These two observations correlate with the immunological data accumulated thus far which shows that antibody raised against the small subunit of the spinach leaf enzyme strongly cross-reacts with the small subunit of the maize endosperm [24], rice endosperm [ 1, 9, 22], Arabidopsis [ 12], and potato tuber [15] enzymes, but does not crossreact well with the large subunits from these plants [15, 24, 22]. In addition, antibody raised against the spinach large subunit exhibits minimal or no reactivity to the large subunit of the maize [24] and potato enzymes [15]. A comparison of the deduced amino acid se-
Fig. 1. Alignment of the deduced large subunit amino acid sequences of the wheat endosperm AGA.7 [ 16], maize endosperm shrunken-2 [4], and potato tuber ADP-glucose pyrophosphorylase. The degree of conservation is indicated in the consensus sequence in which residues conserved in all three sequences are marked with an asterisk. Gaps were introduced to maintain homology.
1091 terest is Tyrl~4 of the E. coli enzyme which has been shown to be involved in substrate binding [ 11 ]. To test whether the phenolic hydroxyl group played a critical role in enzyme catalysis, Kumar et aL [8] replaced Tyrll 4 with Phe in the bacterial enzyme via site-directed mutagenesis. This mutation caused a lowering of the apparent affinity of the enzyme for its substrates but did not appear to be obligatory for catalysis [8]. The altered enzyme also displayed a decreased affinity toward its activator. This region is highly conserved in both the small and large subunits of the potato enzyme; however, in the plant subunits the Tyr is replaced by Phe (Fig. 2). This conservation of a hydrophobic aromatic amino acid at this position may be of importance in coordinating the adenine rings of ATP and ADP-glucose via hydrophobic interactions as suggested by Olive etaL [16].
quence of the small and large potato subunits to the E. coli subunit [3] displayed an overall identity of 37~o and 3 5 ~ respectively (Fig. 2). Despite this low overall homology, the domains which are important for allosteric and catalytic activity of the bacterial enzyme are more conserved. Lys39 and Lys195 of the E. coli sequence have been shown to be protected from phosphopyridoxylation by the activator fructose-l,6Pz and substrate ADP-glucose, respectively, suggesting these two residues are located within or near the allosteric and substrate binding sites [3, 17]. These two regions are highly conserved in both potato subunits (Fig. 2). The high degree of homology in these areas suggests this region may contribute to the stability of the protein conformation and possibly to the regulatory and/or catalytic functions of the plant enzyme. Also of in-
1 Consensus
LS p o t a t o SS p o t a t o E. coll
50
................................................................... V.. ............................................... NKI K P G V A Y S V I T T E N D T Q T V F V MAASIGALKS SPSSNNCINE RRNDSTRAVS SRNLSFSSSH LAGDKLMPVS SLRSQGVRFN VRRSPMIVSP ....................................................................... 101 ** *
**
***
*
LS p o t a t o SS p o t a t o E. coll
GTRL.PLT.K GTKLFPLTS~ GTRLYPLTKK GTRLKDLTN~
Consensus LS p o t a t o SS p o t a t o E. coil
W.FE..K ..... ILA...GD WVFEDAKNKN IENIVVLSGD WLFEEHTVLE YLILA...GD DIIRRYKAEY VVILA...GD
Consensus
RAKPAVP.GG TATPAVPVGG RAKPAVPLGA RAKPAVHFGG
201
**
,
**
,
.YRLIDIP.S CYRLIDIPMS NYRLIDIPVS KFRIIDFALS
** **
*~** LS p o t a t o SS p o t a t o E. cell
,~
,
.IASMGIYVF YIASMGVYVF FIASMGIYVI ..ASMGIYVF
*
**
Consensus
LS p o t a t o SS p o t a t o E. coll
.N ..... N . G GNGVSFGD.G SNMGGYKNEG FFNEE.MND.
FVEVLAAQQ. FVEVLAATQT FVEVLAAQQS FVDLLPAQQR
P..EN..WFQ PGEAGKKWFQ P..ENPDWFQ MKGEN..WYR
,
**
,
*
*
**
*
..DVLL.LI . . . . . P..N... KTDVLLKLLK WSYPTSN... SKDVMLNLLR DKFPGAN... DADYLYELLE EDDRDENSSH
***
,
YT.PR.LPP. YTSPRFLPPT YTQPRYLPPS RTYNESLPPA
GTADAVRQ.L GTADAVRKFI GTADAVRQYL GTADAVTQNL
**
•
,
***
**
.RASAFGIMK SRASDFGLVK KRATAFGLMK EEASAFGVMA
IDE.GR.IEF IDSRGRVVQF IDEEGRIIEF VDENDKTIEF
AEKP.G..L. AF~PKGFDLK AEKPQGEQLQ VF~PANPP..
.... Y W E D I G IFKDYWEDIG LYDGYWEDIG FPLSCVQSDP
T I E . F Y N . . . . . . . . . . . AN T I K S F Y N . . . . . . . . . . . AS TIEAFYN ........... AN DAEP.YWRDV GTLEAYWKAN
300 ,
*
A M . V D T T . . G L .... A K . . P AMQVDTTLVG LSPQDAKKSP AMKVDTTILG LDDKRAKEMP A M P N D P S . . . . . . . . . KSL.
350
**
DFGSE.IP.A DFGSEIIPAA DFGSEVIPGA DFGKDLIPKI
,
ITV.C.P... ITLSCAPAED ITVAALPMDE CTVVCMPVPI
400 •
T..G..VQAY IDDYN.VQAY TSLGMRVQAY TEAGLAYAHP
*
*
*
L.LT...VP. LALTQEF.PK LGITKKPVPD LDLA.SVVPK
450 ***
**
,
**
*
F.FYDR..P] FQFYDPKTPF VSFYDRSAPI LDMYDRNWPI 5OO
.
K ....... D . . . . . . . S . I S .GCVI..C.. .HSVVG.RS. .S.G .... D. K I D N C K I K D A ....... IIS ~ G C F L R D C S V EIISIVGERSR L D C G V E L K D T KMLDADVTD ....... SVIG XGCVIKNCKI HHSVVGLRSC ISEGAIIEDS KFVQDRSGSH GMTLN.SLVS GGCVISG ..... SVVVQSVL FSRVRVNSFC
..MGADYY.T FMMGADYYQT LLMGADYYET NIDSAVLLPE
...... L.A. G . V P I G I G . N ESEIASLLAE GKVPIGIGEN DADRKLLAAK GSVPIGIGKN V~ .............. VGRS
C.IRRCIIDK TKIRKCIIDK CHIKRAIIDK CRLRRCVIDR
550 .
LS p o t a t o SS p o t a t o E. coli
LNRHI.R.Y. LNRHIARTYF LNRHLSRAYA LVQMIQRGWS
250 *
HIYRMDY .... Q.H.E..AD HLYRMDYMEL VQNHIDRNAD HLYRMDYEKF IQAHRETDAD HIYKQDYSRM LIDHVEKGVR
501 Consensus
200
•
.VLTQYNSA. FVLTQYNSAP YVLTQFNSAS GVITQYQSHT
401 ,
. M V S L E . N . . . . L D . . A . . . . . . . ILGGG. D M P R L E R R R A N P K D V A A V I L ...... G G G E KAVSDSQNSQ TCLDPDASRS VLGIILGGGA MVSLEKNDH LMLARQLPLK SVALILAGGR
150 *
NCINS.I.KI NCINSAINKI NCLNSNISKI NCINSGIRRM
301
Consensus
I00
NA.IG.NV.I NAKIGKNVSI NARIGDNVKI ACVIPEGMVI
,
INKDNVQEAA INKDGVQEAD INKDNVQEAA ..GENAEEDA
*
**
R,..GY.I.S RPEEGFYIRS RETDGYFIKS RR..FYRSEE
GIV.V...AL GIIIILEKAT GIVTVIKDAL GIVLVTREML
*
I..G.VI... IRDGTVI... IPSGIVl... RKLGHKQER.
Fig. 2. Amino acid sequence comparison of the potato and E. coli ADP-glucose pyrophosphorylases [3]. The amount of identity between the potato and bacterial subunits is indicated in the consensus sequence. Residues conserved in all three sequences are marked with an asterisk. Residues essential for allosteric and/or catalytic function of the bacterial enzyme and the complimentary residues in the plant subunits are in boldface. The sequences corresponding to the pyfidoxylated peptide associated with the allosteric site of the spinach leaf small subunit is underlined.
1092 Photoaffinity studies have been conducted with the spinach enzyme using 8-N3ADP-glucose [ 23 ]. Incorporation is seen mainly, if not solely, in the large subunit. It will be interesting to see if the area labeled is homologous to the bacterial substrate-binding region. Since the bacterial and plant enzymes catalyze the same reactions their structural dissimilarities most likely reflect their differences in allosteric specificity. Morell et al. [ 13] have shown that pyridoxal phosphate which mimics the effector molecule 3-PGA equally labels both subunits of the spinach enzyme. For the small subunit, the pyridoxal phosphate binding region has been determined and the reactive Lys residue identified [ 13 ]. This Lys residue and surrounding region is identically conserved in the small subunit of potato, rice [1], and spinach [22]. This region is also conserved in the large subunit (Fig. 2). These observations suggest that the allosteric region of the plant enzyme contains an essential peptide sequence located in its C-terminus rather than in its N-terminus as in the bacterial enzyme. The difference in allosteric specificity could also be a result of amino acid changes in other regions of the plant enzyme as suggested earlier by Anderson et al. [ 1 ]. Lee et al. [ 10] and Kumar et al. [7] have shown when Gly336 of the bacterial enzyme is changed to the acidic residue Asp, the bacterial enzyme becomes more refractive towards allosteric activation by fructose-1,6-P2. It is interesting to note that the amino acid at the equivalent position in the potato, rice [1], and spinach [22] small subunits are also negatively charged. It has been speculated that the two plant subunits were originally derived from the same gene based on their low but certain homology and the recent evidence suggesting that the cyanobacterial enzyme, which has higher-plant allosteric specificity, is a homotetramer like the bacterial enzyme [22]. It is quite possible that during evolution, there was a gene duplication in the higher-plant photosynthetic system of the pyrophosphorylase gene and then divergence of the genes to produce two different polypeptides, both of which may be required for optimal activity [22].
Acknowledgements Supported by grants from the National Science Foundation (DMB 86-10319) and U S D A / D o E / N S F Plant Science Center Program (88-372713964) to J.P., the Department of Energy (DEFG06-87ER13699) and Project 0590, College of Agriculture and Home Economics, Washington State University, Pullman, WA 99164-6340 to T.W.O, and a McKnight Foundation Fellowship to P.A.N.
References 1. Anderson JM, Hnilo J, Larson R, Okita TW, Morell M, Preiss J: The encoded primary sequence of a rice seed ADP-glucose pyrophosphorylase subunit and its homology to the bacterial enzyme. J Biol Chem 264: 1223812242 (1989). 2. Anderson JM, Okita TW, Preiss J: Enhancing carbon flow into starch: The role of ADP-glucose pyrophosphorylase. In: Vayda ME, Park WD (eds) The Molecular Biology of the Potato, pp. 159-180. C.A.B. International, Wallingford (1990). 3. Baecker PA, Furlong CE, Preiss J: Biosynthesis of bacterial glycogen: Primary structure of Escherichia coli ADP-glucose synthetase as deduced from the nucleotide sequence of the glgC gene. J Biol Chem 258:5084-5088 (1983). 4. Bhave MR, Lawrence S, Barton C, Hannah C: Identification and molecular characterization of Shrunken-2 c D N A clones of maize. Plant Cell 2:581-588 (1990). 5. du Jardin P, Berhin A: Isolation and sequence analysis of a cDNA clone encoding a subunit of the ADP-glucose pyrophosphorylase of potato tuber amyloplasts. Plant Mol Biol 16:349-351 (1991). 6. Heldt HW, Chon J, Maronde D, Stan Kovic ZS, Walker DA, Kraminer A, Kirk MR, Heber V: Role of orthophosphate and other factors in the regulation of starch formation in leaves and isolated chloroplasts. Plant Physiol 59: 1146-1155 (1977). 7. Kumar A, Ghosh P, Hill MA, Preiss J: Biosynthesis of bacterial glycogen: Determination of the amino acid changes that alter the regulatory properties of a mutant Escherichia coil ADP-glucose synthetase. J Biol Chem 264:10464-10471 (1989). 8. Kumar A, Tanaka T, Lee YM, Preiss J: Biosynthesis of bacterial glycogen: Use of site-directed mutagenesis to probe the role of tyrosine 114 in the catalytic mechanism of ADP-glucose synthetase from Escherichia coli. J Biol Chem 263:14634-14639 (1988). 9. Krishnan HB, Reeves CD, Okita TW: ADP-glucose py-
1093
10.
11.
12.
13.
14.
15.
16.
17.
rophosphorylase is encoded by different mRNA transcripts in leaf and endosperm of cereals. Plant Physiol 81: 642-645 (1986). Lee YM, Kumar A, Preiss J: Amino acid sequence of an Escherichia coli ADP-glucose synthetase allosteric mutant as deduced from the DNA sequence of the glgC gene. Nucl Acids Res 15:10603 (1987). Lee YM, Preiss J: Covalent modification of substratebinding sites of Escherichia coli ADP-glucose synthetase: Isolation and structural characterization of 8-azido-ADPglucose-incorporated peptides. J Biol Chem 261: 10581064 (1986). Lin TP, Caspar T, Somerville C, Preiss J: A starch deficient mutant of Arabidopsis thaliana with low ADPglucose pyrophosphorylase activity lacks one of the two subunits of the enzyme. Plant Physiol 88:1175-1181 (1988). Morell M, Bloom M, Preiss J: Affinity labeling of the allosteric activator site(s) of spinach leaf ADP-glucose pyrophosphorylase. J Biol Chem 263:633-637 (1988). Muller-Rober BT, Kossmann J, Hannah LC, Willmitzer L, Sonnewald U: One of two different ADP-glucose pyrophosphorylase genes from potato responds strongly to elevated levels of sucrose. Mol Gen Genet 224:136-146 (1990). Okita TW, Nakata PA, Anderson JM, Sowokinos J, Morell M, Preiss J: The subunit structure of potato tuber ADP-glucose pyrophosphorylase. Plant Physiol 93: 785790 (1990). Olive MR, Ellis RJ, Schuch WW: Isolation and nucleotide sequences of eDNA clones encoding ADP-glueose pyrophosphorylase polypeptides from wheat leaf and endosperm. Plant Mol Biol 12:525-538 (1989). Parsons TF, Preiss J: Biosynthesis of a bacterial glycogen: Isolation and characterization of the pyridoxal-P al-
18.
19. 20.
21.
22.
23.
24.
25.
losteric activator site and the ADP-glucose-protected pyridoxal-P binding site ofEscherichia coliB ADP-glucose synthase. J Biol Chem 253:7638-7645 (1978). Preiss J: Regulation of ADP-glucose pyrophosphorylase. In: Meister A (ed) Advances in Enzymology and Related Areas of Molecular Biology, vol 46, pp. 317-381. John Wiley, New York (1978). Preiss J: Bacterial glycogen synthesis and its regulation. Annu Rev Microbiol 38:419--458 (1984). Preiss J: Biosynthesis of starch and its regulation. In: Preiss J (ed) The Biochemistry of Plants, vol 14: Carbohydrates, pp. 184-249. Academic Press, San Diego (1988). Preiss J: Biochemistry and molecular biology of starch biosynthesis and its regulation. In: Mifiin BJ (ed) Oxford Survey of Plant Molecular and Cellular Biology, vol 7. Oxford University Press, Oxford, in press. Preiss J, Ball K, Hutney J, Smith-White B, Li L, Okita TW: Regulatory mechanisms involved in the biosynthesis of starch. Pure Appl Chem 63:535-544 (1991). Preiss J, Cress D, Hutney J, Morell M, Bloom M, Okita T, Anderson J: Regulation of starch synthesis: Biochemical and genetic studies. In: Whitaker JR, Sonnet PE (eds) ACS Symposium Series 389 on Biocatalysis in Agricultural Biotechnology, pp. 84-92. American Chemical Society, Washington, DC (1989). Preiss J, Danner S, Summers PS, Morell M, Barton CR, Yang L, Nieder M: Molecular characterization of the Brittle-2 gene effect on maize endosperm ADP-glucose pyrophosphorylase subunits. Plant Physiol 92:881-885 (1990). Preiss J, Ghosh HP, Wittkop J: Regulation of the biosynthesis of starch in spinach chloroplast. In: Goodwin TW (ed) Biochemistry of Chloroplasts, vol 2, pp. 131153. Academic Press, New York (1967).
1089
Update section
Short communication
Comparison of the primary sequences of two potato tuber ADP-glucose pyrophosphorylase subunits Paul A. Nakata, 1 Thomas W. Greene, 2 Joseph M. Anderson, 3 Brian J. Smith-White,4 Thomas W. Okita 3. and Jack Preiss 4 1Program in Biochemistry and Biophysics, 2program in Plant Physiology, and 3Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA (*author for correspondence); 4Department of Biochemistry, Michigan State University, East Lansing, MI 48824, USA Received 3 July 1991; accepted 9 July 1991
Key words: ADP-glucose pyrophosphorylase, potato, starch biosynthesis
Abstract Near-full-length cDNA clones to the small and large subunit of the heterotetrameric potato tuber ADP-glucose pyrophosphorylase have been isolated and characterized. The missing amino terminal sequence of the small subunit has also been elucidated from its corresponding genomic clone. Primary sequence comparisons revealed that each potato subunit had less identity to each other than to their homologous subunit from other plants. It also appeared that the smaller subunit is more conserved among the different plants and the larger subunit more divergent. Amino acid comparisons of both potato tuber sequences to the Escherichia coli ADP-glucose pyrophosphorylase sequence revealed conserved regions important for both catalytic and allosteric function of the bacterial enzyme.
ADP-glucose pyrophosphorylase plays a pivotal role in the biosynthesis of ~l,4-glucans in both bacteria [19] and plants [20, 21, 25]. This tetrameric enzyme mediates the synthesis of the activated glucosyl donor, ADP-glucose, from glucose- 1-P and ATP. The catalytic activity of both the bacterial and plant enzymes are allosterically regulated by small effector molecules which oscillate during normal carbon metabolism in these organisms [18]. In Escherichia coli, ADP-glucose
pyrophosphorylase is activated by fructose-l,6P2 and inhibited by AMP and ADP, whereas the plant enzyme is stimulated by 3-PGA and inhibited by Pi [ 18]. Fluctuation in the 3-PGA/Pi ratio, in response to photosynthesis, has been shown to regulate the transient production of starch in leaf tissue [6]. Whether this allosteric regulation operates in non-photosynthetic tissue is not known. To gain insight into the structure/function relationship of the plant enzyme we have elucidated
The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases.
1090
the heterotetrameric structure of the potato tuber enzyme [15] and have isolated near-full-length c D N A clones to the small subunit as previously reported [2]. Our findings have been corroborated by other groups [5, 14]. Recently we have isolated and characterized a near-full-length c D N A clone to the large subunit of the tuber enzyme and have derived the missing N-terminal coding sequence of the small subunit from its corresponding genomic clone. A comparison of the small and large potato tuber subunits revealed a 52~o amino acid identity overall which is somewhat higher than the 35~o identity observed between the spinach leaf subunits [22]. The potato small subunit showed 8 4 ~ identity to the rice endosperm small subunit [ 1 ] and 93 ~o identity to the spinach leaf small subunit [22] at the primary level. The potato large subunit exhibited 58 ~o and 61~o sequence identity to AGA.7 [16] and shrunken-2 [4] clones, which encode the wheat i
Consensus Wheat Maize Potato
K..... R. . . . $ . . . . . Q. . . . . . R ..NG .......... TT . . . . . T ...... R. R ASPPSESRAP LIb~PORSATR OHOAROGPRR MCNGGRGPPY WTAGVTSAPA RQTPLFSGRP
NSGGKCDFDL A A T F F C S W Y L W G G D M Q F A LALDTNSGPH QIRSCEGDGI DRLEKLSIGG ~KQEKALRNR CFGGRVAATQ QCILTSDCAF ETLHSQTOSS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . NKIKPGVA YSVITTENDT QTVFVDMPRL
.... AOP..V SGGLSDPNEV RKNYADNARV ERRRANPKDV
E.AG..WFQG EAAG..WFRG EPAG.,WFQG E.AGKKWFQG
.
CFNSGINKIF CFNSGINKIF CFNSGINKIF CINSAINKIF
VMTQFNSASL VMTQFNSASL VMSQFNSTSL VLTQYNSAPL
NRHIHRTYLG NRHIHRTYLG NRHIHRTYLE NRHIARTYFG
GGINF.DGSV GGINFTDGSV GGINFADGSV NGVSFGDGFV
VQKHVDDNAD VQKHVDDNAD VQKHVEDDAD VQNHIDRNAD
ITLSCAPV.E ITLSCAPVGE ITISCAPVDE ITLSCAPAED
SRAS..GLVK SRASEYGLVK SRASKNGLVK SRASDFGLVK
I D S . G R W Q F ..EKPKG.DL F D S S G R W Q F SAEQPKGDDL IDHTGRVLOP P.EKPKGADL I D S R G R W Q F A.EKPKGFDL
AAVILGGGTG AAVILGGGTG SAIILGGGTG AAVILGGGEG
TQLFPLTSTR TQLFPLTSTR SOLFPLTSTR TKLFPLTSRT
ATPAVPVGGC ATPAVPIGGC ATPAVPVGGC ATPAVPVGGC
YRLIDIPMSN YRLIDIPMSN YRLIDIPMSN YRLIDIPMSN
TADA.RKFIW TAOAWRKIIW TAOSIRKFIW TADAVRKFIW
VLEDYY..KS VLEDYYKNKS VLEDYYSHKS VFEDAKNKNI
I..IVILSGD IEHILILSGD IDNIVILSGD ENIW.LSGD
QLYRMDYMEL QLYRMDYMEL QLYRMNYMEL HLYRMDYMEL
.AM.VDT.FL EAMKVDTSFL NSMRVETNFL KAMQVDTTLV
Consensus Wheat Maize Potato
PKTPFFTSPR PKTPFFTSFR PKTPFFTAPR PKTPFYTSPR
..AIDDA.K¥ NFAIDDPAKY SYAIDDAQKY GLSPQOAKK$
PYIASMGVYV PYIASMGVyV PYLASMGIYV PYIASMGVyV
FK.DVLL.LL FKRDVLLNLL FKKDALLDLL FRTDVLLKLL
KS.Y..LHDF KSRYAELHDF KSKYTQLHDF KWSYPTSNDF
.LPPTK.DKC YLPPTKSDKC CLPPTQLDKC FLPPTKIDNC
KIK.AIISHG RIKEAIILHG KMRYAFISDG KIKDAIISHG
CFLREC. IEH CFLRECKIEH CLLRECNIEH CFLRDCSVEH
S..G..SRL. TAF...SRLN SVIGVCSRVS $1VGERSRLD
,
• ,i
t
Ittl
***
,
•
t,
,
K.GVQEADRP KEGVQEADRP SKGIQEADHP KDGVQEADRP
EEGYYIRSGI %e41LKNATI. DGTVI EEGYYIRSGI W I Q K N A T I K D G T W EEGYYIRSGI W I L K N A T I N ECLVI EEGFYIRSGI IIILEKATIR DGTVI
***t,t
,
EVLAATQMPG EVLAATQMPG QVLAATQMPE EVLAATQTPG 300
400 GSEILPRA.. GSEILPRALH GSEILPRAVL GSEIIPAAID
DHNVQAYIFT DHNVQAYVFT DHSVQACIFT DYNVQAYIFK
DYWEDIGT~K DYWEDIGTIR GYWEDVGTIK DYWEDIGTIK
SFFDANLALT SFFDANRALC SFFDANLALT SFYNASLALT
EQPPKF.FYD EQPPKFEFYD EQPSKFDFYD QE?PEFQFYD
SG.ELKD..M SGSELKNAMM SGCELKDSVM CGVELKDTFM
MGAD.YETE. MGADSYETED MGADIYETEE MGADYYQTES
E.S.LL.EGK EMSRLMSEGK EASKLLLAGK EIASLLAEGK
VPIGIGENTK VPIGVGENTK VPIGIGRNTK VPIGIGENTK
IRNCIIDMNA ISNCIIDMNA IRNCIIDMNA IRKCIIDKNA
500
450
545
501 RIGKNVVI.N RIGRDWISN RIGKNWITN KZGKNVSIIN
.it**
350
401
Consensus Wheat Maize Potato
t*
250
301
Consensus Wheat Maize Potato
200
150 **********
201 Consensus Wheat Maize Potato
I00
50
............................................. .......................................
101 Consensus Wheat Maize Potato
and maize endosperm large subunits, respectively (Fig. 1). There appears to be less identity between the two subunits from the same species than between similar subunits from the different plants. The identity studies also suggest that the smaller subunits are more conserved among the different plants and the larger subunits more divergent. These two observations correlate with the immunological data accumulated thus far which shows that antibody raised against the small subunit of the spinach leaf enzyme strongly cross-reacts with the small subunit of the maize endosperm [24], rice endosperm [ 1, 9, 22], Arabidopsis [ 12], and potato tuber [15] enzymes, but does not crossreact well with the large subunits from these plants [15, 24, 22]. In addition, antibody raised against the spinach large subunit exhibits minimal or no reactivity to the large subunit of the maize [24] and potato enzymes [15]. A comparison of the deduced amino acid se-
Fig. 1. Alignment of the deduced large subunit amino acid sequences of the wheat endosperm AGA.7 [ 16], maize endosperm shrunken-2 [4], and potato tuber ADP-glucose pyrophosphorylase. The degree of conservation is indicated in the consensus sequence in which residues conserved in all three sequences are marked with an asterisk. Gaps were introduced to maintain homology.
1091 terest is Tyrl~4 of the E. coli enzyme which has been shown to be involved in substrate binding [ 11 ]. To test whether the phenolic hydroxyl group played a critical role in enzyme catalysis, Kumar et aL [8] replaced Tyrll 4 with Phe in the bacterial enzyme via site-directed mutagenesis. This mutation caused a lowering of the apparent affinity of the enzyme for its substrates but did not appear to be obligatory for catalysis [8]. The altered enzyme also displayed a decreased affinity toward its activator. This region is highly conserved in both the small and large subunits of the potato enzyme; however, in the plant subunits the Tyr is replaced by Phe (Fig. 2). This conservation of a hydrophobic aromatic amino acid at this position may be of importance in coordinating the adenine rings of ATP and ADP-glucose via hydrophobic interactions as suggested by Olive etaL [16].
quence of the small and large potato subunits to the E. coli subunit [3] displayed an overall identity of 37~o and 3 5 ~ respectively (Fig. 2). Despite this low overall homology, the domains which are important for allosteric and catalytic activity of the bacterial enzyme are more conserved. Lys39 and Lys195 of the E. coli sequence have been shown to be protected from phosphopyridoxylation by the activator fructose-l,6Pz and substrate ADP-glucose, respectively, suggesting these two residues are located within or near the allosteric and substrate binding sites [3, 17]. These two regions are highly conserved in both potato subunits (Fig. 2). The high degree of homology in these areas suggests this region may contribute to the stability of the protein conformation and possibly to the regulatory and/or catalytic functions of the plant enzyme. Also of in-
1 Consensus
LS p o t a t o SS p o t a t o E. coll
50
................................................................... V.. ............................................... NKI K P G V A Y S V I T T E N D T Q T V F V MAASIGALKS SPSSNNCINE RRNDSTRAVS SRNLSFSSSH LAGDKLMPVS SLRSQGVRFN VRRSPMIVSP ....................................................................... 101 ** *
**
***
*
LS p o t a t o SS p o t a t o E. coll
GTRL.PLT.K GTKLFPLTS~ GTRLYPLTKK GTRLKDLTN~
Consensus LS p o t a t o SS p o t a t o E. coil
W.FE..K ..... ILA...GD WVFEDAKNKN IENIVVLSGD WLFEEHTVLE YLILA...GD DIIRRYKAEY VVILA...GD
Consensus
RAKPAVP.GG TATPAVPVGG RAKPAVPLGA RAKPAVHFGG
201
**
,
**
,
.YRLIDIP.S CYRLIDIPMS NYRLIDIPVS KFRIIDFALS
** **
*~** LS p o t a t o SS p o t a t o E. cell
,~
,
.IASMGIYVF YIASMGVYVF FIASMGIYVI ..ASMGIYVF
*
**
Consensus
LS p o t a t o SS p o t a t o E. coll
.N ..... N . G GNGVSFGD.G SNMGGYKNEG FFNEE.MND.
FVEVLAAQQ. FVEVLAATQT FVEVLAAQQS FVDLLPAQQR
P..EN..WFQ PGEAGKKWFQ P..ENPDWFQ MKGEN..WYR
,
**
,
*
*
**
*
..DVLL.LI . . . . . P..N... KTDVLLKLLK WSYPTSN... SKDVMLNLLR DKFPGAN... DADYLYELLE EDDRDENSSH
***
,
YT.PR.LPP. YTSPRFLPPT YTQPRYLPPS RTYNESLPPA
GTADAVRQ.L GTADAVRKFI GTADAVRQYL GTADAVTQNL
**
•
,
***
**
.RASAFGIMK SRASDFGLVK KRATAFGLMK EEASAFGVMA
IDE.GR.IEF IDSRGRVVQF IDEEGRIIEF VDENDKTIEF
AEKP.G..L. AF~PKGFDLK AEKPQGEQLQ VF~PANPP..
.... Y W E D I G IFKDYWEDIG LYDGYWEDIG FPLSCVQSDP
T I E . F Y N . . . . . . . . . . . AN T I K S F Y N . . . . . . . . . . . AS TIEAFYN ........... AN DAEP.YWRDV GTLEAYWKAN
300 ,
*
A M . V D T T . . G L .... A K . . P AMQVDTTLVG LSPQDAKKSP AMKVDTTILG LDDKRAKEMP A M P N D P S . . . . . . . . . KSL.
350
**
DFGSE.IP.A DFGSEIIPAA DFGSEVIPGA DFGKDLIPKI
,
ITV.C.P... ITLSCAPAED ITVAALPMDE CTVVCMPVPI
400 •
T..G..VQAY IDDYN.VQAY TSLGMRVQAY TEAGLAYAHP
*
*
*
L.LT...VP. LALTQEF.PK LGITKKPVPD LDLA.SVVPK
450 ***
**
,
**
*
F.FYDR..P] FQFYDPKTPF VSFYDRSAPI LDMYDRNWPI 5OO
.
K ....... D . . . . . . . S . I S .GCVI..C.. .HSVVG.RS. .S.G .... D. K I D N C K I K D A ....... IIS ~ G C F L R D C S V EIISIVGERSR L D C G V E L K D T KMLDADVTD ....... SVIG XGCVIKNCKI HHSVVGLRSC ISEGAIIEDS KFVQDRSGSH GMTLN.SLVS GGCVISG ..... SVVVQSVL FSRVRVNSFC
..MGADYY.T FMMGADYYQT LLMGADYYET NIDSAVLLPE
...... L.A. G . V P I G I G . N ESEIASLLAE GKVPIGIGEN DADRKLLAAK GSVPIGIGKN V~ .............. VGRS
C.IRRCIIDK TKIRKCIIDK CHIKRAIIDK CRLRRCVIDR
550 .
LS p o t a t o SS p o t a t o E. coli
LNRHI.R.Y. LNRHIARTYF LNRHLSRAYA LVQMIQRGWS
250 *
HIYRMDY .... Q.H.E..AD HLYRMDYMEL VQNHIDRNAD HLYRMDYEKF IQAHRETDAD HIYKQDYSRM LIDHVEKGVR
501 Consensus
200
•
.VLTQYNSA. FVLTQYNSAP YVLTQFNSAS GVITQYQSHT
401 ,
. M V S L E . N . . . . L D . . A . . . . . . . ILGGG. D M P R L E R R R A N P K D V A A V I L ...... G G G E KAVSDSQNSQ TCLDPDASRS VLGIILGGGA MVSLEKNDH LMLARQLPLK SVALILAGGR
150 *
NCINS.I.KI NCINSAINKI NCLNSNISKI NCINSGIRRM
301
Consensus
I00
NA.IG.NV.I NAKIGKNVSI NARIGDNVKI ACVIPEGMVI
,
INKDNVQEAA INKDGVQEAD INKDNVQEAA ..GENAEEDA
*
**
R,..GY.I.S RPEEGFYIRS RETDGYFIKS RR..FYRSEE
GIV.V...AL GIIIILEKAT GIVTVIKDAL GIVLVTREML
*
I..G.VI... IRDGTVI... IPSGIVl... RKLGHKQER.
Fig. 2. Amino acid sequence comparison of the potato and E. coli ADP-glucose pyrophosphorylases [3]. The amount of identity between the potato and bacterial subunits is indicated in the consensus sequence. Residues conserved in all three sequences are marked with an asterisk. Residues essential for allosteric and/or catalytic function of the bacterial enzyme and the complimentary residues in the plant subunits are in boldface. The sequences corresponding to the pyfidoxylated peptide associated with the allosteric site of the spinach leaf small subunit is underlined.
1092 Photoaffinity studies have been conducted with the spinach enzyme using 8-N3ADP-glucose [ 23 ]. Incorporation is seen mainly, if not solely, in the large subunit. It will be interesting to see if the area labeled is homologous to the bacterial substrate-binding region. Since the bacterial and plant enzymes catalyze the same reactions their structural dissimilarities most likely reflect their differences in allosteric specificity. Morell et al. [ 13] have shown that pyridoxal phosphate which mimics the effector molecule 3-PGA equally labels both subunits of the spinach enzyme. For the small subunit, the pyridoxal phosphate binding region has been determined and the reactive Lys residue identified [ 13 ]. This Lys residue and surrounding region is identically conserved in the small subunit of potato, rice [1], and spinach [22]. This region is also conserved in the large subunit (Fig. 2). These observations suggest that the allosteric region of the plant enzyme contains an essential peptide sequence located in its C-terminus rather than in its N-terminus as in the bacterial enzyme. The difference in allosteric specificity could also be a result of amino acid changes in other regions of the plant enzyme as suggested earlier by Anderson et al. [ 1 ]. Lee et al. [ 10] and Kumar et al. [7] have shown when Gly336 of the bacterial enzyme is changed to the acidic residue Asp, the bacterial enzyme becomes more refractive towards allosteric activation by fructose-1,6-P2. It is interesting to note that the amino acid at the equivalent position in the potato, rice [1], and spinach [22] small subunits are also negatively charged. It has been speculated that the two plant subunits were originally derived from the same gene based on their low but certain homology and the recent evidence suggesting that the cyanobacterial enzyme, which has higher-plant allosteric specificity, is a homotetramer like the bacterial enzyme [22]. It is quite possible that during evolution, there was a gene duplication in the higher-plant photosynthetic system of the pyrophosphorylase gene and then divergence of the genes to produce two different polypeptides, both of which may be required for optimal activity [22].
Acknowledgements Supported by grants from the National Science Foundation (DMB 86-10319) and U S D A / D o E / N S F Plant Science Center Program (88-372713964) to J.P., the Department of Energy (DEFG06-87ER13699) and Project 0590, College of Agriculture and Home Economics, Washington State University, Pullman, WA 99164-6340 to T.W.O, and a McKnight Foundation Fellowship to P.A.N.
References 1. Anderson JM, Hnilo J, Larson R, Okita TW, Morell M, Preiss J: The encoded primary sequence of a rice seed ADP-glucose pyrophosphorylase subunit and its homology to the bacterial enzyme. J Biol Chem 264: 1223812242 (1989). 2. Anderson JM, Okita TW, Preiss J: Enhancing carbon flow into starch: The role of ADP-glucose pyrophosphorylase. In: Vayda ME, Park WD (eds) The Molecular Biology of the Potato, pp. 159-180. C.A.B. International, Wallingford (1990). 3. Baecker PA, Furlong CE, Preiss J: Biosynthesis of bacterial glycogen: Primary structure of Escherichia coli ADP-glucose synthetase as deduced from the nucleotide sequence of the glgC gene. J Biol Chem 258:5084-5088 (1983). 4. Bhave MR, Lawrence S, Barton C, Hannah C: Identification and molecular characterization of Shrunken-2 c D N A clones of maize. Plant Cell 2:581-588 (1990). 5. du Jardin P, Berhin A: Isolation and sequence analysis of a cDNA clone encoding a subunit of the ADP-glucose pyrophosphorylase of potato tuber amyloplasts. Plant Mol Biol 16:349-351 (1991). 6. Heldt HW, Chon J, Maronde D, Stan Kovic ZS, Walker DA, Kraminer A, Kirk MR, Heber V: Role of orthophosphate and other factors in the regulation of starch formation in leaves and isolated chloroplasts. Plant Physiol 59: 1146-1155 (1977). 7. Kumar A, Ghosh P, Hill MA, Preiss J: Biosynthesis of bacterial glycogen: Determination of the amino acid changes that alter the regulatory properties of a mutant Escherichia coil ADP-glucose synthetase. J Biol Chem 264:10464-10471 (1989). 8. Kumar A, Tanaka T, Lee YM, Preiss J: Biosynthesis of bacterial glycogen: Use of site-directed mutagenesis to probe the role of tyrosine 114 in the catalytic mechanism of ADP-glucose synthetase from Escherichia coli. J Biol Chem 263:14634-14639 (1988). 9. Krishnan HB, Reeves CD, Okita TW: ADP-glucose py-
1093
10.
11.
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rophosphorylase is encoded by different mRNA transcripts in leaf and endosperm of cereals. Plant Physiol 81: 642-645 (1986). Lee YM, Kumar A, Preiss J: Amino acid sequence of an Escherichia coli ADP-glucose synthetase allosteric mutant as deduced from the DNA sequence of the glgC gene. Nucl Acids Res 15:10603 (1987). Lee YM, Preiss J: Covalent modification of substratebinding sites of Escherichia coli ADP-glucose synthetase: Isolation and structural characterization of 8-azido-ADPglucose-incorporated peptides. J Biol Chem 261: 10581064 (1986). Lin TP, Caspar T, Somerville C, Preiss J: A starch deficient mutant of Arabidopsis thaliana with low ADPglucose pyrophosphorylase activity lacks one of the two subunits of the enzyme. Plant Physiol 88:1175-1181 (1988). Morell M, Bloom M, Preiss J: Affinity labeling of the allosteric activator site(s) of spinach leaf ADP-glucose pyrophosphorylase. J Biol Chem 263:633-637 (1988). Muller-Rober BT, Kossmann J, Hannah LC, Willmitzer L, Sonnewald U: One of two different ADP-glucose pyrophosphorylase genes from potato responds strongly to elevated levels of sucrose. Mol Gen Genet 224:136-146 (1990). Okita TW, Nakata PA, Anderson JM, Sowokinos J, Morell M, Preiss J: The subunit structure of potato tuber ADP-glucose pyrophosphorylase. Plant Physiol 93: 785790 (1990). Olive MR, Ellis RJ, Schuch WW: Isolation and nucleotide sequences of eDNA clones encoding ADP-glueose pyrophosphorylase polypeptides from wheat leaf and endosperm. Plant Mol Biol 12:525-538 (1989). Parsons TF, Preiss J: Biosynthesis of a bacterial glycogen: Isolation and characterization of the pyridoxal-P al-
18.
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25.
losteric activator site and the ADP-glucose-protected pyridoxal-P binding site ofEscherichia coliB ADP-glucose synthase. J Biol Chem 253:7638-7645 (1978). Preiss J: Regulation of ADP-glucose pyrophosphorylase. In: Meister A (ed) Advances in Enzymology and Related Areas of Molecular Biology, vol 46, pp. 317-381. John Wiley, New York (1978). Preiss J: Bacterial glycogen synthesis and its regulation. Annu Rev Microbiol 38:419--458 (1984). Preiss J: Biosynthesis of starch and its regulation. In: Preiss J (ed) The Biochemistry of Plants, vol 14: Carbohydrates, pp. 184-249. Academic Press, San Diego (1988). Preiss J: Biochemistry and molecular biology of starch biosynthesis and its regulation. In: Mifiin BJ (ed) Oxford Survey of Plant Molecular and Cellular Biology, vol 7. Oxford University Press, Oxford, in press. Preiss J, Ball K, Hutney J, Smith-White B, Li L, Okita TW: Regulatory mechanisms involved in the biosynthesis of starch. Pure Appl Chem 63:535-544 (1991). Preiss J, Cress D, Hutney J, Morell M, Bloom M, Okita T, Anderson J: Regulation of starch synthesis: Biochemical and genetic studies. In: Whitaker JR, Sonnet PE (eds) ACS Symposium Series 389 on Biocatalysis in Agricultural Biotechnology, pp. 84-92. American Chemical Society, Washington, DC (1989). Preiss J, Danner S, Summers PS, Morell M, Barton CR, Yang L, Nieder M: Molecular characterization of the Brittle-2 gene effect on maize endosperm ADP-glucose pyrophosphorylase subunits. Plant Physiol 92:881-885 (1990). Preiss J, Ghosh HP, Wittkop J: Regulation of the biosynthesis of starch in spinach chloroplast. In: Goodwin TW (ed) Biochemistry of Chloroplasts, vol 2, pp. 131153. Academic Press, New York (1967).
