Eur. J. Biochem. 205, 1053- 1059 (1992)
0FEBS 1992
cDNA and gene sequences of wheat chloroplast sedoheptulose-1,7-bisphosphatase reveal homology with fructose-l,6-bisphosphatases Christine A. RAINES', Julie C. LLOYD', Nicola M. WILLINGHAM, Susan POTTS and Tristan A. DYER'
'
Biology Department, University of Essex, Colchester, England Cambridge Laboratory, Centre for Plant Science Research, Norwich, England
(Received January 21, 1992) - EJB 920077
The nucleotide sequence encoding the chloroplast enzyme, sedoheptulose-l,7-bisphosphatase [Sed(l,7)P2ase],was obtained from wheat cDNA and genomic clones. The transcribed region of the Sed(1,7)P2ase gene has eight exons (72 - 507 bp) and seven introns (85 - 626 bp) and encodes a precursor polypeptide of 393 amino acids. Comparison of the deduced amino acid sequence of Sed(1,7)P2ase with those of fructose-l,6-bisphosphatase[Fru(l,6)P2ase] enzymes from a variety of sources reveals 19% identity, rising to 42% if conservative changes are considered. Most importantly, the amino acid residues which form the active site of Fru(l,6)P2ase are highly conserved in the Sed(1,7)P2asemolecule, indicating a common catalytic mechanism. Interestingly, although the activities of both Sed(1,7)P2ase and chloroplast Fru(1 ,6)P2ase are modulated by light via the thioredoxin system, the amino acid sequence motif identified as having a role in this regulation in chloroplast Fru(1 ,6)P2ase is not found in the Sed(l,7)P2ase enzyme.
Sed(1,7)P2ase is increased significantly in light by reduced thioredoxin f (Breazeale et al., 1978; Wirtz et al., 1982); changes in pH and Mg2+ concentration, occurring with light/dark transitions, also modulate Sed(1,7)P2ase activity (Woodrow et al., 1984). In continuous illumination, a finer level of control is exerted by the products, inorganic phosphate (Woodrow et al., 1983) and Sed7P, and by glycerate (a product of photorespiration) and ribulose-1,s-bisphosphate(Schimkat et al., 1990). The chloroplast bisphosphatases occupy strategically important positions in the photosynthetic carbon-reduction pathway, where Sed(l,7)P2ase competes with the starch synthetic pathway for substrate and Fru(1 ,6)P2ase with the sucrose synthetic pathway (Woodrow, 1986). Biochemical analysis of the kinetics of these enzymes has revealed many common properties which include, in addition to light activation, inhibition by both the reaction product and substrate analogue fructose 2,6-bisphosphate [Fru(2,6)P2] and also the ability to utilise each others' substrates (Cadet and Meunier, 1988b; Schimkat et al., 1990). The ubiquitous nature of Fru(l,6)P2ase, with its role in glycolysis and gluconeogenesis, is reflected in the wealth of biochemical data available on this enzyme. Molecular studies have resulted in extensive amino acid sequence information, Correspondence to C. A. Raines, Department of Biology, Univer- including that of pig kidney (Marcus et al., 19821, the wheat sity of Essex, Wivenhoe Park, Colchester, England C 0 4 3SQ (Raines et al., 1988) and spinach (Marcus et al., 1988) Fux: + 44206 873416. chloroplast enzymes, and also in the determination of the Abbreviotions. Sed(1,7)P2ase, sedoheptulose-1.7-bisphosphatase; crystallographic structure of the pig kidney enzyme to a Fru(l,6)Pzase. fructose-l,6-bisphosphatase;Sed7P, sedoheptulose 7-phosphate; Fru(2,6)P2, fructose 2,6-bisphosphate; Fru6P. fructose resolution of 0.28 nm (Ke et al., 1989a, b, 1990, 1991a, b). In contrast to this, Sed(l,7)P2ase (like ribulose-1,s-bisphosphate 6-phosphate. Enzymes. Sedoheptulose-l,7-bisphosphatase(EC 3.1.3.37); chlo- carboxylase and phosphoribulokinase) is unique to the roplast fructose-l,6-bisphosphatase (EC 3.1.3111). photosynthetic carbon-reduction cycle; consequently, much
The photosynthetic carbon-reduction cycle is the primary pathway of carbon fixation which in higher plants takes place in the stroma of chloroplasts. This cycle is autocatalytic; in addition to producing substrates for starch and sucrose biosynthesis, it must regenerate the carbon dioxide acceptor, ribulose 1,s-bisphosphate. A balance between the regenerative and export functions is critical, and this is believed to be achieved by mechanisms which regulate the catalytic activity of a number of key enzymes of the cycle. In addition to the constraints imposed by ribulose-1,5-bisphosphate carboxylase on the overall rate of carbon flow, three further enzymes, sedoheptulose-1,7-bisphosphatase[Sed(l,7)P2ase], fructose1,6-bisphosphatase [Fru(l ,6)P2ase]and phosphoribulokinase, have been proposed to have prominent roles in regulating the flow of intermediates (Woodrow and Berry, 1988). The reactions catalysed by these enzymes are essentially irreversible and their activities are stimulated significantly by light (Wirtz et al., 1982). Sed(1,7)P2ase catalyses the dephosphorylation of Sed(1,7)Pz, forming sedoheptulose 7-phosphate (Sed7P) and inorganic phosphate, and this is an essentially irreversible reaction which commits intermediates to the regenerative part of the photosynthetic carbon-reduction cycle. The activity of
1054 less information on the molecular characteristics of Sed(1,7)P2ase is available. However, Sed(l,7)P2ase has been purified from wheat (Woodrow and Walker, 1982), maize (Nishizawa and Buchanan, 1981) and spinach (Cadet et al., 1987), and evidence to date suggests that it has a dimeric structure with a monomer molecular mass of 35 - 38 kDa. In this paper we present the first information on the primary structure of Sed(1,7)Pzase, derived from wheat cDNA and genomic nucleotide sequences. These data have been used to make comparisons with Fru(l,6)Pzase from diverse sources, and wc discuss the structural similarities which this has revealed between the bisphosphatases. Most significantly, we show that the active-site structure determined by crystallographic studies for ~ ~,6)P2ase ( 1is conserved in Sed(1,7)P2ase. MATERIALS AND METHODS Plant material Seeds of Triticum aestivum (cv Mercia) were grown for 7 days in a Fisons controlled environmental chamber, with a day length of 16 h, at 23°C. Etiolated plants were grown for 7 days in sealed boxes in a darkened environmental chamber. Library screening Initially, Sed(l,7)Pzase cDNA clones were isolated from a previously constructed wheat cDNA library (Raines et al., 1988), using the method of Young and Davis (1983). The filters were screened using maize Sed(1,7)P2ase polyclonal antibodies (a gift from B. B. Buchanan; see Nishizawa and Buchanan, 1981) for 20 h. Positive clones were identified using perixodase-conjugated goat anti-(rabbit IgG) antibodies (BioRad) and the chromogenic peroxidase substrate chloro-lnaphthol. A clone (S1.l) isolated in this way was labelled using random primers (Feinberg and Vogelstein, 1983) and used both to rescreen the original cDNA library and to probe a wheat genomic library (Lloyd et al., 1991). Filters were hybridised in 6 x NaCl/Cit (NaCl/Cit: 0.15 M NaCl and 0.015 M citrate, pH 7.0), Denhardt's, 0.1% SDS and 50 pg/ml herring sperm DNA for 24 h at 65"C, washed extensively in 2 x NaCl/Cit and 0.1% SDS at room temperature followed by 2 x NaCl/Cit and 1% SDS at 65°C for 15 min then exposed to X-ray film. Positively hybridising phages were purified to homogeneity and the Sed(l,7)Pzase inserts subcloned into the plasmid vector PUBS1 [a pUC19 derivative containing the polylinker region of pBluescript (Stratagene)] for sequencing. The direction of the coding strand of S1.l cDNA was determined by subcloning into M13mp18 and M13mp19, producing single-stranded radiolabelled probes by primer extension, as described by Huttly et al. (1988). These probes were hybridized independently to Northern blots of wheat-leaf mRNA (see below) and a signal obtained from one only, which was the strand complementary to the mRNA species detected . DNA sequencing The DNA sequence of the Sed(l,7)Pzase cDNA clones and selected restriction-enzyme fragments from genomic clones were determined on both strands. Plasmid subclones were digested with exonuclease I11 after cutting with appropri-
Fig. 1. Northern blot analysis of Sed(l,7)PzasemRNA in 7-day-old kht(L)- and dark(D)-grown wheat leaves. Equal amounts ( 5 k%) of pob(A)-richRN.4 were probed with the S9.2 Sed(l,7)P2ase(SBPase) cDNA and, as a control, with a wheat cytoplasmic phosphoglycerate kinase (PGKase)cDNA (Longstaff et al., 1989).
ate restriction enzymes (Henikoff, 1984) to create a set of nested deletions. These clones were then sequenced by the dideoxy-chain-termination method using a double-stranded plasmid technique and Sequenase (Murphy and Ward, 1990). Primer extension and S1 nuclease analysis S1 nuclease analysis was performed using a 314-bp fragment extending from an upstream SmaI site to a Not1 site at position 13 in Fig. 3. This was subcloned into M13mp18 and M13mp19 to produce single-stranded probes which were used for S1 nuclease analysis, as described by Huttly et al. (1988). To augment the results from S1 nuclease analysis, primer extension was also performed, using methods from Sambrook et al. (1989). Initially a 20-nucleotide primer, complementary to bases 212-231 in Fig. 3, then also a 34-nucleotide primer, complementary to bases 16-18 in Fig. 3 , were end-labelled, and 10 pmol annealed to 2 pg wheat-leaf poly(A)-rich RNA using standard techniques (Sambrook et al., 1989) before extension with reverse transcriptase (Amersham cDNA synthesis kit). Products were detected on a 6% sequencing gel, with a ladder of sequencing reactions of the Sed(l,7)P2ase gene primed using the same oligonucleotide alongside as a marker. RNA analysis Poly(A)-rich RNA was isolated as described by Baulcombe and Buffard (1983). The RNA (5 pg) was electrophoresed in 1.2% formaldehyde/Mops gels using standard procedures (Sambrook et al., 1989), blotted onto nylon membrane (Hybond N', Amersham) and probed with 32P-labelled DNA (Feinberg and Vogelstein, 1983) according to the rnanufacturers instructions. The size of mRNA was estimated using RNA markers (Gibco BRL). RESULTS Isolation and characterisationof cDNA and genomic clones encoding wheat Sed(l,7)PZase The polyclonal antisera raised against maize Sed(1,7)P,ase (Nishizawa and Buchanan, 1981) was used to screen a wheatleaf cDNA expression library. These antibodies have previously been shown to have a high specificity for Sed(1,7)P,ase and did not cross-react with maize chloroplast, cytosolic or mammalian Fru(l,6)P2ase enzymes (Nishizawa and
1055
A
scale=200 bp
s 3.3
S 9.2
s 1.1
E
B
X H
v v
L
X H
S
E
S
v v ,
v
s s
A 5.2
-
E
scale=l Kb
-
A1 3
exonsize(bp) lntron size (bp)
I
II
111
w
v
VI
VII
169
138
186
82
222
101
72
85
122
88
456
94
103
vlll
507 626
Fig. 2. Restriction maps of wheat Sed(l,7)P2asecDNA and genomic clones. E, EcoRI; H, HindIII; S, SstI; X, Xbal. Open bars represent coding regions and stippled bars represent non-coding regions of the cDNA. (A) Positions of the partial cDNA clones, S 1 . l , S9.2 and S3.3, within the Sed(l.7)P2ase mRNA. (B) Organisation of the Sed(l,7)P2asegene and position in two wheat genomic DNA inserts from 5.2 and 1.3.
Buchanan, 1981). On Western blots, these antibodies react with only one band in crude tissue extracts from wheat, maize, tobacco and Arabidupsis (data not shown). Of 1.5-3.0 x lo5 recombinants screened, three positives were obtained and found by cross-hybridization to fall into two classes. One of the positives, S1.l (500 bp). was selected for further analysis on the basis of RNA blot studies, which showed that it recognised an RNA species which was both light-induced (Fig. l), as would be expected for an enzyme involved in the photosynthetic carbon-reduction cycle (Raines et al., 1991), and of approximately the predicted size. Since S1.l was too small to contain the full-length cDNA, it was used as a probe to rescreen both the cDNA library and a wheat genomic library. Ten further cDNA recombinants were isolated of which S9.2 was about 70 bp longer than S1.l. The sequence of S9.2 was determined, and the insert used to ascertain the coding strand. as described in Materials and Methods. The results of these experiments showed that the cDNA clones terminated at an EcoRI site within the coding sequence, presumably due to ineffective methylation of such sites during the cloning procedure. To obviate this cloning defect, the six genomic clones isolated using the S1.l probe were restriction enzyme mapped. Also, a genomic fragment, distal to the internal EcoRI site, was isolated to probe the cDNA library for the missing sequences of the 3’ end. The genomic clones were of two classes, represented by 1.3 and 5.2 (Fig. 2). and the probe used was a terminal EcuRI fragment from 5.2. The longest of the cDNA clones isolated. S3.3, extended from the internal EcoRI site to the poly(A) tail. The relationship between the cDNA and genomic clones is illustrated in Fig. 2. Nucleotide sequence The nucleotide sequence encoding the wheat Sed( 1,7)P2ase enzyme was obtained from two partial cDNA clones (S9.2 and S3.3) and two genomic clones (Ll.3 and 15.2). The combined 1282 bp of sequence obtained from the cDNA clones comprises 1057 bp of coding sequence and a 224-nucleotide 3’ untranslated region terminating in a poly(A) tail. The majority of the genomic sequence was obtained from 15.2. However. this clone ended 23 codons before the termination codon
in exon VIII, and the remaining 3’ sequence is from E.1.3. No differences were found between the cDNA and genomic nucleotide sequences where these overlapped, and seven introns (I1 - VIII; Fig. 2) were found to lie within the coding region. Genomic sequence upstream of the truncated cDNA was obtained and both primer extension and S1 nuclease mapping used to determine that the region of transcription initiation is between position -74 and -71 (Fig. 3; see Materials and Methods for descriptions of probes and primers used). This analysis revealed a further intron of 85 bp, as shown in Fig. 2. The nucleotide sequence of the wheat Sed(l,7)P2ase gene is presented in Fig. 3. The coding region of this gene therefore has eight exons in the range 72 - 507 bp and seven introns in the range 85 - 626 bp. Derived amino acid sequence Comparison of the derived amino acid sequence shown in Fig. 3 with a range of Fru(l,6)P2ases (see Table 1) suggests that this clone does not encode Fru(l,6)P2ase and this, taken together with the high specificity of the antibodies (Nishizawa and Buchanan, 1981), gives a strong indication that this clone encodes wheat Sed(1,7)P2ase. The gene shown in Fig. 3 encodes a protein of 393 amino acids which would give a polypeptide with a molecular mass greater than 35 - 38 kDa predicted for a monomer subunit of Sed(l,7)P2ase(Nishizawa and Buchanan, 1981; Cadet et al., 1987). We suggest that, since the N-terminal region of this polypeptide has features in common with chloroplast transit peptides (von Heijne, 1989), it is synthesised in a precursor form with a leader sequence which directs the protein to the chloroplast, where it is cleaved to form one of the functional subunits of the Sed(l,7)P2ase holoenzyme. The exact position of the cleavage site for removal of the transit peptide is unknown because as yet no N-terminal sequence for any Sed(1,7)P2aseenzyme is available. However, analysis of a range of chloroplast transit sequences has shown that the C-terminus tends to have increased numbers of Arg residues, and the region from - 3 to + 1 of the cleavage site tends not to have Ser, Gly, Asp, Asn or Pro, but has increased numbers of Ala (von Heijne et al., 1989). This data leads us
1056 1416 CCACCTTCGTAGTTGCCCTCMGGACTGCCCCGGCACACACGMTTCCTT
-05 TCTTCTGAMTCTTMGCTCCGTAGCCTCTCTCTCCTCGTCAGCGCCGGC -35 CAGACCACGCACCGGCAGCCAGCCAGCCAGCAGCMTGGACACCGTCGCG H
E
T
V
T L
16 GCCGCCGGCTACGCGCACGGGGCCGCCACGCGCTCCCCGGCGTGCTGCGC A
A
G
Y
A
H
G
A
A
T
R
S
P
A
C
C
M
S
F
S
P
S
Y
R
P
R
P
L
R
A
Y
T
A
R
T
A S
T
S
F
Y
G
E
F
P
A
G
R
K
A
A
S
R
A
A
L
T
T
R
C
A
I
G
K
P
D
K
Y
L
I
R
L
F
L
T
K
A
L
I
C
M
G
E
A
M
516 GAGGACGATCGCCTTCMGGTCCGGACGGCCTCCTGCGGCGGCACGGCCT T
I
A
F
K
V
R
T
A
S
C
G
G
T
A
C
566 GCGTCAACTCCTTCGGCGACGAGCAGCTCGCCGTCGACATGCTCGCCGAC V
N
S
F
G
D
E
P
L
A
V
D
M
L
A
D
616 AAGCTCCTCTTCGAGGTGAGACGGCCGTGAGCCAGCGCTTTCCATCCGTG K
L
L
F
E
666 AGTGCACGCCTCGTCCCGTCACGGTTCCTCACCTCGCTTGTATATATATG 71 6 CAGGCGTTGGAGTACTCCCATGTGTGCMGTACGCGTGCTCTGAGGAAGT A
L
E
Y
S
H
V
C
K
Y
A
C
S
E
E
E
L
P
D
M
G
G
P
V
E
D
TCTCGACGAGATGGTTMGCAGGAAGACGATCGCCTTATGTTTTMCCGT GTTCTTGGCAGMCCGCGTGCCATTCCTGCGCCT~TACATAGTGGTAC
1916 1966 2016 2066 21 16 2166 2216 2266 2316 2366 2416 2466
TAGTCTGCTGCTGMCAAAACACATGTCCACCTTCTCACCTTGAGAGCAC
S
S
I
V
D
T
F
N
S
F
1316 ACCGTGGGAACCATCTTCGGCGTCTGGCCCGGCGACMGCTGACCGGCGT T
V
G
T
I
F
G
V
U
P
G
D
K
L
T
G
V
1366 CACCGGCGGTGACCAGGTTGCTGCCGCCATGGGCATCTACGGCCCTCGCA T
G
G
D
P
V
A
A
A
M
G
I
Y
G
P
R
L
U
P
H
V
K
D
T
T
S
I
G
E
G
S
P
G
N
L
R
A
T
F
D
N
P
D
Y
V
N
Y
Y
V
K
E
K
Y
T
L
R
G
G
H
V
P
D
V
N
P
MTCACAAATGTTATTCAGAGTTTCAGACCCAGCCCTACAAACACCTGCA TTTGTMMTCACTTGCATTGCATGCGTGGAGCMTCAAATGCCATGGCA CTAGTTCTTCAGTACCGATCGGTGATCTCATTCATCATCACGMTAGTGC CGGTTCCACTCAGCATCGMCAGGGCCATGATCCATCTGTCATGTCMTT CTACTCCTGCGCCTTTGGCGCCAGAGAGGACGACMTCTTGGAGTCGAGG CAATGTMCTTTTCTGAGAACAACAATTTGGCTGGGTGCTCGCAGCTGTG AGTGCCACCMCTCTTGTTCCACCGCGCGCCCATGTGGTCGACCATCGAG
CCTGTGCCGGCCTCTGCTTTMGCMGGCCATGCATGCTGTCGATCGACC GGCACGGCGCGTACTAGTGCACCMCTTTGCTTTGCTTCCGTATACACTC CATTGCATGCAGAGATTCAAATCTTTCGTTTTAGCGTTATTACTCGTGTG CATGCATGCATGAGMGAGAGGGGTGTATGTTTGAT~TGGTGCGTGCA
lGCATGCAGATCATCGTGAAGGAWUGGGCATCTTCACWUCGTGACGTC
T F
A L
V
L
I
V
K
E
K
G
I
F
T
Y
V
T
S
K
A
K
L
R
L
L
F
E
V
A
P
L
G
I
E
K
A
C
G
H
S
S
D
G
K
P
S
D
K
V
I
S
V
L
D
E
R
T
P
V
A
Y
T
S
K
N
E
I
I
R
F
E
E
T
L
Y
G
S
S
2716 CCAGACTCGCCGCCAGCGCCACCGTCGGCGCCACCGCCTMTAATCTTCT R
1266 GTGTGGCGTTCGACCCCCTTGACGGCTCCAGCATCGTGGACACCMCTTC G
T
G
CTGMTTATGATCGGCTCGGGTTTTCGGTCGCGTCTCGCAGGCGGATTCA
D
F
2616 GTGCTGGACAAGGTCATCTCCGTCCTGGACGAGCGGACCCAGGTGGCCTA
TACGTAGCTCATTTTAGCTGGCGTTCACGAGATGCAGAMTTTTGGCTTT
L
E
2566 GGTTCTTGATAGAGMGGCCGGCGGGCACAGCAGCGACGGCMGCAGTCG
TATACCAGTGGAACMCGTATCAGTACATGTAACCATGGCGTCTTCTTGG
P
H
2666 CGGCTCCMGMCCAGATCATCCGCTTCGAGGAGACCCTCTACGGCTCCT
ATTCCACTCATAGGAACCATCCACTCGCCAGCAGCTACATGCACTGTAGC
D
T
2516 GCCGACGGCGMGGCGMGCTGCGGCTGCTGTTCGAGGTGGCGCCGCTGG
TGTAGTACTGGGCAGAGTCCAGTGCCACACTACACCAGCMCACCACTGC
F
G
K
P
916 966 1016 1066 1116 1166 1216
A
P
1066 TATTCAGTTATTACMTTTACACMGTCATATGTTTCTCGCATACTTAGG
G
066
V
C
G
I
816 G C A T A T C C A T C T T G T C A C G A T A A A T M T C A G C C A G G M T C A A A G C C C ~
G
F
V
766 CCCCGAGCTGCAGGACATGGGTGGCCCGGTCGMGGTACTTTACATAACT P
E
L
466 ACGCCGGACMGMCCTCATCAGGCTGCTGATCTGCATGGGGGAGGCGAT
R
M
Y
E
D
1666 ATGACAAGGTGGTACCAG~TACTGTTTGGTTGTTGCTTCTCTGCACGGCT
L
E
K
1816 TACACCGGAGGAATGGTCCCTGATGTCMCCAGGTACATACATCMCMC
366 GTCCCCTGCGCGTCGCCGGTGTCACGTGMGCTAACGGTGTCTGTGCACT 416 TTTTGGTTTTTTTTTTTGCGTGGTGGCAGGAGGAGTTCCTGACCAAGGCG
T
D
D
316 CAGCCTGGTTCGTGCCCTGCACCCGCCACCAGTATACATGCTCTGGTCTA S
L
1716 CTAAACTGATGTAGCAACAGTTGTMTCAGMCCTMCMCTGCAAMTG 1766 TATCCTGTCAGCTTCTCMCTACTATGTGMGGACAAGTACACTCTGCGT
P
266 TCCMGGCGGCCAGCCGGGCGGCGCTCACCACCCGCTGCGCGATCGGCGA S
A
K
216 AGTCGCTGCGGGCGMCACGGCGAGGACGTCGTTCCCGGCGGGGCGGCAG S
V
1616 GMGATGTTCTCCCCTCGCMTCTGAGGGCCACGTTCCACMCCCTGATT
166 TGGGCAATGCGTGCGGCAGGCTGCCAGGCCGGCGACCTCGTTCTACGGCG A
L
K
116 TATCCAACATGTGGTCAGGCGTCAACCGTTTTTGTTTTGCTGATGCCGGA A
V
1516 T G G A G C T T C T G T A T G C G T G T G A C A G T G C T M T C ~ G G T C T A C T A C ~ T 1566 GCTGCAGGTAAATGGCAGCATGTCMG~CACCACCAGCATCGGAGMGG
A
66 CGCCATGTCCTTCTCGCAGTCCTACAGGCCCAAGGTGTGTMTGTTAGGA A
F
1466 C T C C T C G A C G M G G T T T T T A C T T C T C T T C T C A A T C M G T C C C C M T G G M
A
2766 2016 2066 2916 2966 3016 3066 31 16
L
A
A
S
A
T
V
G
A
T
A
*
TCTTCTTCTTTTTTTACGCCTTTCTCAACCTG~TTTAAACMGCTGT CCGTTATACAGTCTGMGCATGCTGTTGTCTTGTGATCATCC~CTGGT MTACAGTCGGCAGACCGGCTTGCGTACGTCTGCCGCCCTTGTCTCCACT GCGAGGCCATTGTTGTACGTACACAGTCGTGCGTTTGTGCATGGCATTGG
CMCACTTGTGTCATCACATGTCTGCGTGTCTMCCTMGCAGGCMCGT GATCTCCGATCTCTGCACCTTTGCTCCTCTCCCTCTCTCTCGCACACACA GGACTGTCACGCMCACTCCCGATGTTTCCACCAGCTACTACCAGCCAGG CCGCATGCMCAGTCCACGGTATGGGGT
Fig. 3. Nucleotide sequence of genomic clones coding for wheat Sed(l,7)P2ase. The deduced amino acid sequence is given below the nucleotide sequence. Nucleotides are numbered at the ends of lines, with the first letter of the initiating ATG codon as 1.
to suggest tentatively that the cleavage site of wheat Sed(l,7)P2ase may be in the region Arg59-Cys66, giving a processed subunit polypeptide of 328 - 334 amino acids, with a molecular mass of around 36 kDa, which is in agreement with previous data for maize (Nishizawa and Buchanan, 1981) and spinach (Cadet et al., 1987) Sed(l,7)P2ase. Comparison of Sed(l,7)P2ase and Fru(l,6)Pzase amino acid sequences
In order to determine the relationship between Sed(1,7)P2ase and Fru(1,6)P2ase, pairwise comparisons were
made between the derived amino acid sequence of wheat Sed(l.7)P2ase and Fru(l,6)P2ase from a variety of sources (Table 1) using the Wisconsin GAP programme (Devereux et al., 1984). This reveals that 29-32% of residues in Sed(1,7)P2aseare identical to those in all of the Fru(1 ,6)P2ases examined (including the plant cytosolic enzyme) and, if conservative changes are taken into account, this similarity rises to around 52%. Alignment of the wheat Sed(l,7)P2ase amino acid sequence with those of the wheat chloroplast and pig kidney Fru(l,6)P2asesis shown in Fig. 4. Although all three enzymes have identical residues in only 19% of the positions (42%
1057 Table 1. Identity and similarity between wheat Sed(l,7)Pzase and a variety of Fru(l,6)Pzase amino acid sequences. Sources of sequences: wheat Sed(1,7)P2ase, this work; wheat chloroplast Fru(l,6)P2ase, Raines et al. (1988); spinach cytosolic Fru(l,6)P2ase, Ladror et al. (1990); pig Fru(l,6)P2ase, Marcus et al. (1982); Saccharomyces cerevisiae Fru(l,6)P2ase, Rogers et al. (1988); E. coli Fru(l,6)P2ase, Hamilton et al. (1988); Rhodobacrer sphoeroides Fru(l,6)P2ase, Gibson et al. (1990). The alignments used to generate these values were made using the Wisconsin GAP programme.
Enzyme
Source
Identity with wheat Sed (1,7)P2ase
Fru( 1,6)P2ase from wheat chloroplast
spinach cytosol
pig
S. cerevisiae
E. coli
R. sphaero ides
%
Sed( I ,7)P2ase
wheat
-
29.6
29.8
31.8
28.1
31.8
30.0
Fru(l,6)P2ase
when chloroplast spinach cytosol Pig S . cerevisiae E. coli R . sphoreroides
51.5 52.7 53.7 52.0 53.3 53.0
-
50.0
69.5 63.1 65.7 64.6 59.0
-
44.7 51.8
73.9 71.1 62.8 52.0
-
41 .O 48.6 47.6
68.0 60.5 57.0
67.8 58.0
46.9 44.0 42.6 43.0 58.0
35.0 32.0 34.0 32.0 37.0
wfbp
1
-
40
20
WDTASAPAPAAARKRSSYDMITLTTWLLKQEQEGVIDNEMTIVLSSISTACKQIASLVQRA
PfbP
:: I : I::: :I :: I : 1 1 I: I 11 : : --------QSKAASRAALTTRCAIGDSLEEFLTKATPDKNLIRLLICMGEAMRTIAFKVRTA : I I :: : I :: 1 1 I( : )::I: 1 1 1 -----------TDQAAFDTNIVTLTRFVMEQGRKARGTGEMTQLLNSLCTAWISTAVRKA
wfbp
PISNLTGVQGATNVQGEDQXKLVISNEVFSNCLRWSGRTGVIASEEEDVPVAVEESYSGNY
wsbp
80
60
wsbp PfbP wfbp
I
1::l
I
:I::::
100
I :I
.. ..
Ill
I : I : I 1::I :1:1:: I: : 1 1 :I1 1 : GIAHLYGIAGSTNVTGDPVKKLDVLSNDLVINVLKSSFATCVLVTEEDXNAIIVEPEKRGKY S----
:
:: :
CGGTACVNSFGDEQLAVDMLADKLLFEALEYSHVCKYACSEEVPELQDMGGPVEGGF
+
120
**
140
160
wsbp
IWFDPLDGSSNIDAAVSTGSIFGIYSPSDECHIGDDATLDEVTQMCIVMlCQPGSNLLAAG I:: : I I : I I I I I I I I I :I : 1:lIl:: I II SVAFDPLDGSSIVDTNFTVGTIFGVWP--------- GDKLTGVT---------- GGDQVAAA
PfbP
WCFDPLDGSSNIDCLVSIGTIFGIYWOJSTDEPSEKDAL------------ QPERNLVAAG
I I I I I I I I I :I 4
::l:lll::
I I
Ill:
:
*+
200
180
220
wfbp
YCMYSSSVIFVLTIGTGVYVFTLDPMYGEFVLTQEKQIPKSGKIYSFNEGNYALWDDKL-K
wsbp
MGIYGPRTTFWALKDCPGTHEFLLLDEGKWQHVKDTTSIGTFDNP-:I:: I :I::: : I ::I I II
PfbP
.. ..
:I:::
:I::
.. ..
ll::l
I
I1
YALYGSA~LVLAMVNGVNC~DPAIGEFILVDRNVKIKKKGSIYSINEG-YAKEFDPAIT
+
240
260
+
280
wf bp
XYMDSLKEPGTSGKPYSARYIGSLVGDFHRTMLYG-GIYGYPSDQKSKNGKLRUYECAPMS
wsbp
DYDKLVNY~EK--YTLRYTGGMVPDVNQIIVKEKGIFTLRLLFEVAPLG
PfbP wfbp wsbp pfbp
-
I : ) I I::I I :: :: 1 1 : : : : : ~11::~ :: : 1 : 1 1 I : I ) 1 1 : : :: 1 1 1 : : I:I1III:I I:: EYIERKXFPPDNSAPYGARYVGSMVADVHRTLVYG-GIFMYPANKKSPKGKLRLLYECNPMA ** *+* + + * * I :I
::
~
~
~
:
300 320 FIAEQAGGKGSDGHQRVLDIlrIPTAVHQRVPLYVGSVEEEKFLSSE-----------I: I : J I I 1 : : :::I : :I: 1 : :I FLIEKAGGHSSDGKQSVLDKTSVLDERTQVAYGSKNEIISATVGATA : : : ::~ ~ I ~ I : ~ ~: 1 1 ::: : 1
:Ill
Il::ll
111
11
:::I:
YVMEKAGGLATTGKEAVLDIVPTDIHQRAPIILGSPEDVTELLEIYQKHA------------
Fig. 4. Amino acid sequence alignment of wheat chloroplast Sed(l,7)PZase(wsbp) with wheat chloroplast Fru(l,6)Pzase (wfbp; Raines et al., 1988) and pig kidney Fru(l,6)Pzase (pfbp; Marcus et al., 1982). The amino acids are numbered according to the pig kidney sequence and the transit peptides of the chloroplast enzymes are not shown. Identical residues are indicated by solid lines and conservative changes with a dotted lines. * or denotes residues implicated in Fru(l,6)P2ase substrate binding (Ke et al., 1989b) which are either present ( * ) or absent (+) in Sed(l,7)P2ase.
+
including conservative changes), it is obvious from this alignment that, rather than being evenly dispersed throughout the sequence, there are distinct regions where the sequence is
highly conserved. These regions correspond to blocks of identity which have been found to exist in all Fru(l,6)P2ases studied to date, and contain the residues which have been
~
1058 Table 2. Comparisonof amino acid residues in Fru(l,6)Pzaseimplicated in substrate binding with residues at identical positions in wheat Sed(l,7)P2ase. + indicates the presence of the same residue as that found in the pig sequence. - indicates that a gap was introduced into the alignment at the corresponding position. The figures in brackets are the N B R F point acceptable values. Non-conservative changes score negative values and conscrvative changes either zero or positive values. ~~
Substrate part
Fru(l,6)P2ase from Pig
Wheat Sed( 1,7)P2ase
wheat ~
6-phosphate
Sugar group
( (2)-phosphate
Arg243 Tyr244 Am212 Tyr215 Tyr264 Lys269 Lys274 Glu280 Arg276 Gly246 Ser241 Met248 Asp121 Lys274 Lys274 Arg216 Ser124
+ + + + + + + + + + + Leu (4) + + + + +
+ + Leu ( -2) Asn (-4) -
+ + + + GlY (1) + + + + + +
implicated in binding of the substrate analogue Fru(2,6)P2 (Ke et al., 1989a, b) and the reaction product fructose 6-phosphate (Fru6P; Ke et al., 1990, 1991a, b). Active site residues The overall degree of similarity between the bisphosphatases is sufficient to allow us to make use of the structural information available for pig kidney Fru( 1 ,6)P2ase. The location of the active site was determined from crystallographic studies using the substrate analogue /?-~-Fru(2,6)P,(Ke et al., 1989a, b) and with the product Fru6P (Ke et al., 1990,1991 a, b). If we consider the 19 residues implicated in substrate binding to pig kidney Fru(l,6)P2ase (Ke et al., 1989b) then Sed(1,7)P2ase contains 12 of these residues. The information presented in Table 2 shows that the amino acids which would bind to the sugar moeity are highly conserved, as are the residues which would interact with the phosphate at C1 which is removed by the enzyme. However, the residues which interact with the stable phosphate at C6 in pig and wheat Fru(l,6)P2ases are not so well conserved in Sed(1,7)P2ase and may account for the different substrate specificities of the two enzymes. The residues Aspll8, Asp121 and Glu280, which bind Mg2+ close to the active site in Fru(l,6)P2ase, are all conserved in Sed(l,7)P2ase. Sed(l,7)Plase activation In vivo, the activities of Sed(l,7)P2ase (Breazeale et al., 1978; Wirtz et al., 1982; Cadet and Meunier, 1988a) and chloroplast Fru(1 ,6)P2ase (Zimmerman et al., 1976) are modulated by reduced thioredoxin f which is generated by the light reactions of photosynthesis. The wheat and spinach chloroplast Fru(1 ,6)P2ases have been shown to have a unique insertion sequence (at position
Leu1 53 in the pig sequence), containing two cysteine residues which may be involved in the light regulation of these enzymes (Raines et al., 1988; Marcus et al., 1988). The alignment in Fig. 4 reveals that the wheat chloroplast Sed(1,7)P2ase contains no such insertion and, indeed, none of the cysteines present in Fru(1 ,6)P2ase and Sed(1,7)P2ase align with one another, suggesting that the light-activation region in these two enzymes is different, despite their overall similarities. Sed(t ,7)P2ase has seven cysteines, comprising two pairs and three single residues. One pair of cysteines is separated by four intervening residues (CGGTAC), a structural arrangement which is found for the redox-active groups of a number of enzymes, including mercuric reductase, glutathione reductase, lipoamide dehydrogenase (Fox and Walsh, 1983) and two ribonucleotide reductases (Lin et al., 1987). A subset of these, whch includes Fru(1 ,6)P2ase but not Sed(l,7)P2ase, also share greater similarity of the residues flanked by the cysteine pair (Marcus et al., 1988).
DISUSSION Sed(l,7)Pzase is unique to photosynthetic organisms, where it functions in the photosynthetic carbon-reduction cycle. This enzyme shares a number of functional similarities with the chloroplast Fru(l,6)P2ase, therefore one might expect these enzymes to have structural features in common. However, to date there is very little information on the molecular characteristics of Sed(1,7)P,ase, and both immunological studies and amino acid composition data have emphasised the differences between Fru(1 ,6)P2ase and Sed(1,7)P2ase (Cadet and Meunier, 1988a). Our sequence comparisons (Fig. 4) have shown that there is appreciable similarity between the wheat Sed(1,7)P2ase and Fru(1 ,6)P2ases from diverse sources. The most interesting aspect of our analysis is that the active-site residues shown by crystallographic analysis of pig kidney Fru(l,6)P2ase (Ke et al., 1989a, b, 1990, 1991a, b) are conserved in Sed(1,7)P2ase. Our results support biochemical data which indicate that the chloroplast Sed(1,7)P2ase molecule has one active site which is capable of binding either Sed(1,7)Pz or Fm(l ,6)P2 (Cadet and Meunier, 1988b). These authors also suggested that negative charges would be present near the active site of Sed(1,7)P2ase. The alignment in Fig. 4 shows that all the residues which form a negatively charged pocket, comprising one wall of the binding site of Fru(l,6)P2ase (Glu97 and Glu98, Asp118 and Aspl21, Glu280), are also found in wheat Sed(l,7)P2ase. With the availability of the wheat cDNA clone encoding this enzyme it will now be possible to test these hypotheses using a combination of in vitro mutagenesis and expression of the protein in Esckerichia coli. The similarity which exists between Sed(l,7)P2ase and chloroplast Fru(1,6)P2ase raises the question of the mechanism by which these enzymes evolved. Sed(l,7)P2ase might have arisen from a common bisphosphatase progenitor before the development of oxygenic photosynthesis, or diverged more recently from a bifunctional Sed(1,7)P2ase/Fru(l,6)P2ase enzyme such as that found in cyanobacteria (Gerbling et al., 1986). Although unlikely, convergent evolution of these enzymes cannot be ruled out. More information will be needed on the occurrence and properties of Sed(l,7)Pzase in prokaryotes to indicate which of these explanations is more likely. We have compared the structure of the wheat Sed(1,7)P2ase gene (seven introns) with those available for Fru(l,6)P2ase, namely the wheat chloroplast (three introns;
1059 Lloyd et al., 1991) and rat liver genes (six introns; ElMaghrdbi et al., 1991). No introns are found at identical positions in these genes, although intron 111 in the wheat Sed(l,7)P2ase gene is within four codons of intron I of wheat chloroplast Fru(l,6)P2ase, and introns IV and V of are one and four codons away from the positions of introns I1 and IV of rat liver Fru(l,6)P2ase, respectively. It seems unlikely that these observations are of any evolutionary significance, as the two Fru(1 ,6)P2ase genes themselves show least correspondence of intron positions. Evidence from biochemical studies, including control analysis, has shown that the activity of Sed(1,7)P2aseis tightly regulated. which has led to the belief that this enzyme plays a key role in controlling the flow of intermediates through the photosynthetic carbon reduction cycle (Woodrow, 1986; Woodrow and Berry, 1988; Pettersson and Ryde-Pettersson, 1989). The isolation of sequences encoding wheat Sed(1,7)P2aseshould now allow this to be tested using transgenic plants with altered amounts of this enzyme. We would like to thank Professor B. B. Buchanan for the Sed(1 ,7)P2ase antibodies. This work was supported by the Royal Society (J.C.L.). an AFRC PMB grant (S.P.) and a SERC studentship (N.M.W.). C. A. Raines and J. C. Lloyd both made equal contributions to this work.
REFERENCES Baulcombe. D.C. & Buffard, D. (1983) Planta (Berl.) 157,493-501. Breazeale, V. D.. Buchanan, B. B. & Wolosiuk, R.A. (1978) Z. Naturforsch. 33c, 521 - 528. Cadet, F.. Meunier, J.-C. & Ferte. N. (1987) Biochem. J . 241, 71 -74. Cadet, F. & Meunier. J.-C. (1988a) Biochem. J. 253,243-248. Cadet. F. & Mcunier, J.-C. (1988b) Biochem. J. 253, 249-254. Devereux. J.. Haeberli, P. & Smithies, 0. (1984) Nucleic Acids Res. 12,387-395. El-Maghrabi, M. R., Lange, A. J., Kummel, L. & Pilkis, S. J. (1991) J . Biol. Cliem. 266. 2115-2120. Fcinberg, A.P. & Vogelstein. B. (1983) Anal. Biochem. 132, 6-13. Fox. B. S. & Walsh. C. T. (1983) Biuchemistry 22,4082-4088. Gerbling. K.-P.. Steup, M. & Latzko, E. (1986) Plant Physiol. 80, 716-720. Gibson. J. L., Chen, J. H., Tower, P. A. & Tabita, F. R. (1990) Biochemistry 2Y, 8085 - 8093. Hamilton. W. D. 0.. Harrison, D. A. & Dyer, T. A. (1988) Nucleic Acids Rex 16. 8707. Henikoff. S . (1984) Gene (Amst.) 28, 351 -359. Huttly, A . K., Martienssen, R. A. & Baulcombe, D. C. (1988) Mol. Gen. Genet. 214, 232 - 240. Ke, H.. Thorpe. C. M., Seaton. B. A., Marcus, F. & Lipscomb, W. N. (1989a) Proc. Natl Acad. Sri. U S A 86, 1475-1479.
Ke. H., Thorpe. C. M., Seaton, B. A., Lipscomb, W. N. & Marcus, F. (1989b) J. Mol. Biol. 212, 513-539.Ke, H., Zhang, Y. & Lipscomb, W. N. (1990) Pruc. Natl Acud Sci. USA 87, 52435247. Ke, H., Zhang, Y.. Liang, J.-Y. & Lipscomb, W.N. (19Yla) Proc. Natl Acud Sci. USA 88, 2989 - 2993. Ke. H., Liang, J.-Y., Zhang, Y. & Lipscomb. W.N. (1991 b) Biuchemistry 30,441 2 - 4420. Ladror, U. S., Latshaw, S. P. & Marcus. F. (1990) Eur. J . Biochem. 189, 89 - 94. Lin, A.-E. I. , Ashley, G. W. & Stubbe, J. (1987) Biochemistry 26, 6905 - 6909. Lloyd, J. C., Raines, C. A., John, U. P. & Dyer, T. A. (1991) Mol. Gen. Genet. 225. 209 - 216. Longstaff, M., Raines, C. A,, McMorrow, E. M., Bradbeer, J . W. & Dyer, T. A. (1989) Nucleic Acids Res. 17, 6569-6580. Marcus, F., Edelstein, I., Reardon, I. & Heinrikson, R. L. (1982) Proc. Natl Acad. Sci. USA 79, 7161 -7165. Marcus, F., Moberly, L. & Latshaw, S. P. (1988) Proc. Natl Acad. Sci. U S A 85, 5379 - 5383. Murphy, G. J. P. & Ward, E. S. (1990) in Nucleic acids sequencing a practical approach (Howe, C. J. & Ward, E. S. eds) pp. 99 115, IRL Press, Oxford. Nishizawa, A. N. & Buchanan, B. B. (1981) J . Biol.Cheni. 256,61196126. Petterson, G. & Ryde-Petterson, U. (1989) Eur. J . Biuchem. 186,683 687. Raines, C. A,, Lloyd, J. C., Longstaff, M., Bradley, D. & Dyer, T. A. (1988) Nucleic acid^ Res. 16, 7931 -7942. Rogers, D. T., Hiller, E., Mitsock. L. & Orr. E. (1988) J. B i d . Chem. 263,6051 -6057. Sambrook, J.. Fritsch, E. F. & Maniatis, T. (1989) Molecular cloning: a Iuboratory manual, 2nd edn, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Schimkat, D., Heincke, D. & Heldt, H. W. (1990) Planta (Berl.) 181, 97-103. Wirtz, W., Stitt, . & Heldt, H. W. (1982) FEBS Lett. 142, 223-226. Woodrow, I. E. &Walker, D. A. (1Y82) Arch. Biochem. Biophys. 216, 416 -422. Woodrow, 1. E.. Murphy, D. J. & Walker, D. A. (1983) Eur. J . Biochem. 132, 121 - 123. Woodrow, I. E., Murphy, D. J. & Latzko. E. (1984) J . Biol. Chem. 259, 3791 -3795. Woodrow, I. E. & Berry. J. A. (1988) Annu. Rev. Plant Physiol. Plunt Mol. Biol. 39, 533 - 594. Woodrow, T. E. (1986) Biochim. Biophys. Acta 851, 181 - 192. Von Heijne, G., Steppuhn, J. & Hermann, R. G. (1989) Eur. J . Biochem. 180, 535 - 545. Young, R. A. & Davis, K. W. (1983) Science 222, 778-782. Zimmermann, G.. Kelly, G. J. & Latzko, E. (1976) Eur. J. Biochem. 70, 361 - 367.
0FEBS 1992
cDNA and gene sequences of wheat chloroplast sedoheptulose-1,7-bisphosphatase reveal homology with fructose-l,6-bisphosphatases Christine A. RAINES', Julie C. LLOYD', Nicola M. WILLINGHAM, Susan POTTS and Tristan A. DYER'
'
Biology Department, University of Essex, Colchester, England Cambridge Laboratory, Centre for Plant Science Research, Norwich, England
(Received January 21, 1992) - EJB 920077
The nucleotide sequence encoding the chloroplast enzyme, sedoheptulose-l,7-bisphosphatase [Sed(l,7)P2ase],was obtained from wheat cDNA and genomic clones. The transcribed region of the Sed(1,7)P2ase gene has eight exons (72 - 507 bp) and seven introns (85 - 626 bp) and encodes a precursor polypeptide of 393 amino acids. Comparison of the deduced amino acid sequence of Sed(1,7)P2ase with those of fructose-l,6-bisphosphatase[Fru(l,6)P2ase] enzymes from a variety of sources reveals 19% identity, rising to 42% if conservative changes are considered. Most importantly, the amino acid residues which form the active site of Fru(l,6)P2ase are highly conserved in the Sed(1,7)P2asemolecule, indicating a common catalytic mechanism. Interestingly, although the activities of both Sed(1,7)P2ase and chloroplast Fru(1 ,6)P2ase are modulated by light via the thioredoxin system, the amino acid sequence motif identified as having a role in this regulation in chloroplast Fru(1 ,6)P2ase is not found in the Sed(l,7)P2ase enzyme.
Sed(1,7)P2ase is increased significantly in light by reduced thioredoxin f (Breazeale et al., 1978; Wirtz et al., 1982); changes in pH and Mg2+ concentration, occurring with light/dark transitions, also modulate Sed(1,7)P2ase activity (Woodrow et al., 1984). In continuous illumination, a finer level of control is exerted by the products, inorganic phosphate (Woodrow et al., 1983) and Sed7P, and by glycerate (a product of photorespiration) and ribulose-1,s-bisphosphate(Schimkat et al., 1990). The chloroplast bisphosphatases occupy strategically important positions in the photosynthetic carbon-reduction pathway, where Sed(l,7)P2ase competes with the starch synthetic pathway for substrate and Fru(1 ,6)P2ase with the sucrose synthetic pathway (Woodrow, 1986). Biochemical analysis of the kinetics of these enzymes has revealed many common properties which include, in addition to light activation, inhibition by both the reaction product and substrate analogue fructose 2,6-bisphosphate [Fru(2,6)P2] and also the ability to utilise each others' substrates (Cadet and Meunier, 1988b; Schimkat et al., 1990). The ubiquitous nature of Fru(l,6)P2ase, with its role in glycolysis and gluconeogenesis, is reflected in the wealth of biochemical data available on this enzyme. Molecular studies have resulted in extensive amino acid sequence information, Correspondence to C. A. Raines, Department of Biology, Univer- including that of pig kidney (Marcus et al., 19821, the wheat sity of Essex, Wivenhoe Park, Colchester, England C 0 4 3SQ (Raines et al., 1988) and spinach (Marcus et al., 1988) Fux: + 44206 873416. chloroplast enzymes, and also in the determination of the Abbreviotions. Sed(1,7)P2ase, sedoheptulose-1.7-bisphosphatase; crystallographic structure of the pig kidney enzyme to a Fru(l,6)Pzase. fructose-l,6-bisphosphatase;Sed7P, sedoheptulose 7-phosphate; Fru(2,6)P2, fructose 2,6-bisphosphate; Fru6P. fructose resolution of 0.28 nm (Ke et al., 1989a, b, 1990, 1991a, b). In contrast to this, Sed(l,7)P2ase (like ribulose-1,s-bisphosphate 6-phosphate. Enzymes. Sedoheptulose-l,7-bisphosphatase(EC 3.1.3.37); chlo- carboxylase and phosphoribulokinase) is unique to the roplast fructose-l,6-bisphosphatase (EC 3.1.3111). photosynthetic carbon-reduction cycle; consequently, much
The photosynthetic carbon-reduction cycle is the primary pathway of carbon fixation which in higher plants takes place in the stroma of chloroplasts. This cycle is autocatalytic; in addition to producing substrates for starch and sucrose biosynthesis, it must regenerate the carbon dioxide acceptor, ribulose 1,s-bisphosphate. A balance between the regenerative and export functions is critical, and this is believed to be achieved by mechanisms which regulate the catalytic activity of a number of key enzymes of the cycle. In addition to the constraints imposed by ribulose-1,5-bisphosphate carboxylase on the overall rate of carbon flow, three further enzymes, sedoheptulose-1,7-bisphosphatase[Sed(l,7)P2ase], fructose1,6-bisphosphatase [Fru(l ,6)P2ase]and phosphoribulokinase, have been proposed to have prominent roles in regulating the flow of intermediates (Woodrow and Berry, 1988). The reactions catalysed by these enzymes are essentially irreversible and their activities are stimulated significantly by light (Wirtz et al., 1982). Sed(1,7)P2ase catalyses the dephosphorylation of Sed(1,7)Pz, forming sedoheptulose 7-phosphate (Sed7P) and inorganic phosphate, and this is an essentially irreversible reaction which commits intermediates to the regenerative part of the photosynthetic carbon-reduction cycle. The activity of
1054 less information on the molecular characteristics of Sed(1,7)P2ase is available. However, Sed(l,7)P2ase has been purified from wheat (Woodrow and Walker, 1982), maize (Nishizawa and Buchanan, 1981) and spinach (Cadet et al., 1987), and evidence to date suggests that it has a dimeric structure with a monomer molecular mass of 35 - 38 kDa. In this paper we present the first information on the primary structure of Sed(1,7)Pzase, derived from wheat cDNA and genomic nucleotide sequences. These data have been used to make comparisons with Fru(l,6)Pzase from diverse sources, and wc discuss the structural similarities which this has revealed between the bisphosphatases. Most significantly, we show that the active-site structure determined by crystallographic studies for ~ ~,6)P2ase ( 1is conserved in Sed(1,7)P2ase. MATERIALS AND METHODS Plant material Seeds of Triticum aestivum (cv Mercia) were grown for 7 days in a Fisons controlled environmental chamber, with a day length of 16 h, at 23°C. Etiolated plants were grown for 7 days in sealed boxes in a darkened environmental chamber. Library screening Initially, Sed(l,7)Pzase cDNA clones were isolated from a previously constructed wheat cDNA library (Raines et al., 1988), using the method of Young and Davis (1983). The filters were screened using maize Sed(1,7)P2ase polyclonal antibodies (a gift from B. B. Buchanan; see Nishizawa and Buchanan, 1981) for 20 h. Positive clones were identified using perixodase-conjugated goat anti-(rabbit IgG) antibodies (BioRad) and the chromogenic peroxidase substrate chloro-lnaphthol. A clone (S1.l) isolated in this way was labelled using random primers (Feinberg and Vogelstein, 1983) and used both to rescreen the original cDNA library and to probe a wheat genomic library (Lloyd et al., 1991). Filters were hybridised in 6 x NaCl/Cit (NaCl/Cit: 0.15 M NaCl and 0.015 M citrate, pH 7.0), Denhardt's, 0.1% SDS and 50 pg/ml herring sperm DNA for 24 h at 65"C, washed extensively in 2 x NaCl/Cit and 0.1% SDS at room temperature followed by 2 x NaCl/Cit and 1% SDS at 65°C for 15 min then exposed to X-ray film. Positively hybridising phages were purified to homogeneity and the Sed(l,7)Pzase inserts subcloned into the plasmid vector PUBS1 [a pUC19 derivative containing the polylinker region of pBluescript (Stratagene)] for sequencing. The direction of the coding strand of S1.l cDNA was determined by subcloning into M13mp18 and M13mp19, producing single-stranded radiolabelled probes by primer extension, as described by Huttly et al. (1988). These probes were hybridized independently to Northern blots of wheat-leaf mRNA (see below) and a signal obtained from one only, which was the strand complementary to the mRNA species detected . DNA sequencing The DNA sequence of the Sed(l,7)Pzase cDNA clones and selected restriction-enzyme fragments from genomic clones were determined on both strands. Plasmid subclones were digested with exonuclease I11 after cutting with appropri-
Fig. 1. Northern blot analysis of Sed(l,7)PzasemRNA in 7-day-old kht(L)- and dark(D)-grown wheat leaves. Equal amounts ( 5 k%) of pob(A)-richRN.4 were probed with the S9.2 Sed(l,7)P2ase(SBPase) cDNA and, as a control, with a wheat cytoplasmic phosphoglycerate kinase (PGKase)cDNA (Longstaff et al., 1989).
ate restriction enzymes (Henikoff, 1984) to create a set of nested deletions. These clones were then sequenced by the dideoxy-chain-termination method using a double-stranded plasmid technique and Sequenase (Murphy and Ward, 1990). Primer extension and S1 nuclease analysis S1 nuclease analysis was performed using a 314-bp fragment extending from an upstream SmaI site to a Not1 site at position 13 in Fig. 3. This was subcloned into M13mp18 and M13mp19 to produce single-stranded probes which were used for S1 nuclease analysis, as described by Huttly et al. (1988). To augment the results from S1 nuclease analysis, primer extension was also performed, using methods from Sambrook et al. (1989). Initially a 20-nucleotide primer, complementary to bases 212-231 in Fig. 3, then also a 34-nucleotide primer, complementary to bases 16-18 in Fig. 3 , were end-labelled, and 10 pmol annealed to 2 pg wheat-leaf poly(A)-rich RNA using standard techniques (Sambrook et al., 1989) before extension with reverse transcriptase (Amersham cDNA synthesis kit). Products were detected on a 6% sequencing gel, with a ladder of sequencing reactions of the Sed(l,7)P2ase gene primed using the same oligonucleotide alongside as a marker. RNA analysis Poly(A)-rich RNA was isolated as described by Baulcombe and Buffard (1983). The RNA (5 pg) was electrophoresed in 1.2% formaldehyde/Mops gels using standard procedures (Sambrook et al., 1989), blotted onto nylon membrane (Hybond N', Amersham) and probed with 32P-labelled DNA (Feinberg and Vogelstein, 1983) according to the rnanufacturers instructions. The size of mRNA was estimated using RNA markers (Gibco BRL). RESULTS Isolation and characterisationof cDNA and genomic clones encoding wheat Sed(l,7)PZase The polyclonal antisera raised against maize Sed(1,7)P,ase (Nishizawa and Buchanan, 1981) was used to screen a wheatleaf cDNA expression library. These antibodies have previously been shown to have a high specificity for Sed(1,7)P,ase and did not cross-react with maize chloroplast, cytosolic or mammalian Fru(l,6)P2ase enzymes (Nishizawa and
1055
A
scale=200 bp
s 3.3
S 9.2
s 1.1
E
B
X H
v v
L
X H
S
E
S
v v ,
v
s s
A 5.2
-
E
scale=l Kb
-
A1 3
exonsize(bp) lntron size (bp)
I
II
111
w
v
VI
VII
169
138
186
82
222
101
72
85
122
88
456
94
103
vlll
507 626
Fig. 2. Restriction maps of wheat Sed(l,7)P2asecDNA and genomic clones. E, EcoRI; H, HindIII; S, SstI; X, Xbal. Open bars represent coding regions and stippled bars represent non-coding regions of the cDNA. (A) Positions of the partial cDNA clones, S 1 . l , S9.2 and S3.3, within the Sed(l.7)P2ase mRNA. (B) Organisation of the Sed(l,7)P2asegene and position in two wheat genomic DNA inserts from 5.2 and 1.3.
Buchanan, 1981). On Western blots, these antibodies react with only one band in crude tissue extracts from wheat, maize, tobacco and Arabidupsis (data not shown). Of 1.5-3.0 x lo5 recombinants screened, three positives were obtained and found by cross-hybridization to fall into two classes. One of the positives, S1.l (500 bp). was selected for further analysis on the basis of RNA blot studies, which showed that it recognised an RNA species which was both light-induced (Fig. l), as would be expected for an enzyme involved in the photosynthetic carbon-reduction cycle (Raines et al., 1991), and of approximately the predicted size. Since S1.l was too small to contain the full-length cDNA, it was used as a probe to rescreen both the cDNA library and a wheat genomic library. Ten further cDNA recombinants were isolated of which S9.2 was about 70 bp longer than S1.l. The sequence of S9.2 was determined, and the insert used to ascertain the coding strand. as described in Materials and Methods. The results of these experiments showed that the cDNA clones terminated at an EcoRI site within the coding sequence, presumably due to ineffective methylation of such sites during the cloning procedure. To obviate this cloning defect, the six genomic clones isolated using the S1.l probe were restriction enzyme mapped. Also, a genomic fragment, distal to the internal EcoRI site, was isolated to probe the cDNA library for the missing sequences of the 3’ end. The genomic clones were of two classes, represented by 1.3 and 5.2 (Fig. 2). and the probe used was a terminal EcuRI fragment from 5.2. The longest of the cDNA clones isolated. S3.3, extended from the internal EcoRI site to the poly(A) tail. The relationship between the cDNA and genomic clones is illustrated in Fig. 2. Nucleotide sequence The nucleotide sequence encoding the wheat Sed( 1,7)P2ase enzyme was obtained from two partial cDNA clones (S9.2 and S3.3) and two genomic clones (Ll.3 and 15.2). The combined 1282 bp of sequence obtained from the cDNA clones comprises 1057 bp of coding sequence and a 224-nucleotide 3’ untranslated region terminating in a poly(A) tail. The majority of the genomic sequence was obtained from 15.2. However. this clone ended 23 codons before the termination codon
in exon VIII, and the remaining 3’ sequence is from E.1.3. No differences were found between the cDNA and genomic nucleotide sequences where these overlapped, and seven introns (I1 - VIII; Fig. 2) were found to lie within the coding region. Genomic sequence upstream of the truncated cDNA was obtained and both primer extension and S1 nuclease mapping used to determine that the region of transcription initiation is between position -74 and -71 (Fig. 3; see Materials and Methods for descriptions of probes and primers used). This analysis revealed a further intron of 85 bp, as shown in Fig. 2. The nucleotide sequence of the wheat Sed(l,7)P2ase gene is presented in Fig. 3. The coding region of this gene therefore has eight exons in the range 72 - 507 bp and seven introns in the range 85 - 626 bp. Derived amino acid sequence Comparison of the derived amino acid sequence shown in Fig. 3 with a range of Fru(l,6)P2ases (see Table 1) suggests that this clone does not encode Fru(l,6)P2ase and this, taken together with the high specificity of the antibodies (Nishizawa and Buchanan, 1981), gives a strong indication that this clone encodes wheat Sed(1,7)P2ase. The gene shown in Fig. 3 encodes a protein of 393 amino acids which would give a polypeptide with a molecular mass greater than 35 - 38 kDa predicted for a monomer subunit of Sed(l,7)P2ase(Nishizawa and Buchanan, 1981; Cadet et al., 1987). We suggest that, since the N-terminal region of this polypeptide has features in common with chloroplast transit peptides (von Heijne, 1989), it is synthesised in a precursor form with a leader sequence which directs the protein to the chloroplast, where it is cleaved to form one of the functional subunits of the Sed(l,7)P2ase holoenzyme. The exact position of the cleavage site for removal of the transit peptide is unknown because as yet no N-terminal sequence for any Sed(1,7)P2aseenzyme is available. However, analysis of a range of chloroplast transit sequences has shown that the C-terminus tends to have increased numbers of Arg residues, and the region from - 3 to + 1 of the cleavage site tends not to have Ser, Gly, Asp, Asn or Pro, but has increased numbers of Ala (von Heijne et al., 1989). This data leads us
1056 1416 CCACCTTCGTAGTTGCCCTCMGGACTGCCCCGGCACACACGMTTCCTT
-05 TCTTCTGAMTCTTMGCTCCGTAGCCTCTCTCTCCTCGTCAGCGCCGGC -35 CAGACCACGCACCGGCAGCCAGCCAGCCAGCAGCMTGGACACCGTCGCG H
E
T
V
T L
16 GCCGCCGGCTACGCGCACGGGGCCGCCACGCGCTCCCCGGCGTGCTGCGC A
A
G
Y
A
H
G
A
A
T
R
S
P
A
C
C
M
S
F
S
P
S
Y
R
P
R
P
L
R
A
Y
T
A
R
T
A S
T
S
F
Y
G
E
F
P
A
G
R
K
A
A
S
R
A
A
L
T
T
R
C
A
I
G
K
P
D
K
Y
L
I
R
L
F
L
T
K
A
L
I
C
M
G
E
A
M
516 GAGGACGATCGCCTTCMGGTCCGGACGGCCTCCTGCGGCGGCACGGCCT T
I
A
F
K
V
R
T
A
S
C
G
G
T
A
C
566 GCGTCAACTCCTTCGGCGACGAGCAGCTCGCCGTCGACATGCTCGCCGAC V
N
S
F
G
D
E
P
L
A
V
D
M
L
A
D
616 AAGCTCCTCTTCGAGGTGAGACGGCCGTGAGCCAGCGCTTTCCATCCGTG K
L
L
F
E
666 AGTGCACGCCTCGTCCCGTCACGGTTCCTCACCTCGCTTGTATATATATG 71 6 CAGGCGTTGGAGTACTCCCATGTGTGCMGTACGCGTGCTCTGAGGAAGT A
L
E
Y
S
H
V
C
K
Y
A
C
S
E
E
E
L
P
D
M
G
G
P
V
E
D
TCTCGACGAGATGGTTMGCAGGAAGACGATCGCCTTATGTTTTMCCGT GTTCTTGGCAGMCCGCGTGCCATTCCTGCGCCT~TACATAGTGGTAC
1916 1966 2016 2066 21 16 2166 2216 2266 2316 2366 2416 2466
TAGTCTGCTGCTGMCAAAACACATGTCCACCTTCTCACCTTGAGAGCAC
S
S
I
V
D
T
F
N
S
F
1316 ACCGTGGGAACCATCTTCGGCGTCTGGCCCGGCGACMGCTGACCGGCGT T
V
G
T
I
F
G
V
U
P
G
D
K
L
T
G
V
1366 CACCGGCGGTGACCAGGTTGCTGCCGCCATGGGCATCTACGGCCCTCGCA T
G
G
D
P
V
A
A
A
M
G
I
Y
G
P
R
L
U
P
H
V
K
D
T
T
S
I
G
E
G
S
P
G
N
L
R
A
T
F
D
N
P
D
Y
V
N
Y
Y
V
K
E
K
Y
T
L
R
G
G
H
V
P
D
V
N
P
MTCACAAATGTTATTCAGAGTTTCAGACCCAGCCCTACAAACACCTGCA TTTGTMMTCACTTGCATTGCATGCGTGGAGCMTCAAATGCCATGGCA CTAGTTCTTCAGTACCGATCGGTGATCTCATTCATCATCACGMTAGTGC CGGTTCCACTCAGCATCGMCAGGGCCATGATCCATCTGTCATGTCMTT CTACTCCTGCGCCTTTGGCGCCAGAGAGGACGACMTCTTGGAGTCGAGG CAATGTMCTTTTCTGAGAACAACAATTTGGCTGGGTGCTCGCAGCTGTG AGTGCCACCMCTCTTGTTCCACCGCGCGCCCATGTGGTCGACCATCGAG
CCTGTGCCGGCCTCTGCTTTMGCMGGCCATGCATGCTGTCGATCGACC GGCACGGCGCGTACTAGTGCACCMCTTTGCTTTGCTTCCGTATACACTC CATTGCATGCAGAGATTCAAATCTTTCGTTTTAGCGTTATTACTCGTGTG CATGCATGCATGAGMGAGAGGGGTGTATGTTTGAT~TGGTGCGTGCA
lGCATGCAGATCATCGTGAAGGAWUGGGCATCTTCACWUCGTGACGTC
T F
A L
V
L
I
V
K
E
K
G
I
F
T
Y
V
T
S
K
A
K
L
R
L
L
F
E
V
A
P
L
G
I
E
K
A
C
G
H
S
S
D
G
K
P
S
D
K
V
I
S
V
L
D
E
R
T
P
V
A
Y
T
S
K
N
E
I
I
R
F
E
E
T
L
Y
G
S
S
2716 CCAGACTCGCCGCCAGCGCCACCGTCGGCGCCACCGCCTMTAATCTTCT R
1266 GTGTGGCGTTCGACCCCCTTGACGGCTCCAGCATCGTGGACACCMCTTC G
T
G
CTGMTTATGATCGGCTCGGGTTTTCGGTCGCGTCTCGCAGGCGGATTCA
D
F
2616 GTGCTGGACAAGGTCATCTCCGTCCTGGACGAGCGGACCCAGGTGGCCTA
TACGTAGCTCATTTTAGCTGGCGTTCACGAGATGCAGAMTTTTGGCTTT
L
E
2566 GGTTCTTGATAGAGMGGCCGGCGGGCACAGCAGCGACGGCMGCAGTCG
TATACCAGTGGAACMCGTATCAGTACATGTAACCATGGCGTCTTCTTGG
P
H
2666 CGGCTCCMGMCCAGATCATCCGCTTCGAGGAGACCCTCTACGGCTCCT
ATTCCACTCATAGGAACCATCCACTCGCCAGCAGCTACATGCACTGTAGC
D
T
2516 GCCGACGGCGMGGCGMGCTGCGGCTGCTGTTCGAGGTGGCGCCGCTGG
TGTAGTACTGGGCAGAGTCCAGTGCCACACTACACCAGCMCACCACTGC
F
G
K
P
916 966 1016 1066 1116 1166 1216
A
P
1066 TATTCAGTTATTACMTTTACACMGTCATATGTTTCTCGCATACTTAGG
G
066
V
C
G
I
816 G C A T A T C C A T C T T G T C A C G A T A A A T M T C A G C C A G G M T C A A A G C C C ~
G
F
V
766 CCCCGAGCTGCAGGACATGGGTGGCCCGGTCGMGGTACTTTACATAACT P
E
L
466 ACGCCGGACMGMCCTCATCAGGCTGCTGATCTGCATGGGGGAGGCGAT
R
M
Y
E
D
1666 ATGACAAGGTGGTACCAG~TACTGTTTGGTTGTTGCTTCTCTGCACGGCT
L
E
K
1816 TACACCGGAGGAATGGTCCCTGATGTCMCCAGGTACATACATCMCMC
366 GTCCCCTGCGCGTCGCCGGTGTCACGTGMGCTAACGGTGTCTGTGCACT 416 TTTTGGTTTTTTTTTTTGCGTGGTGGCAGGAGGAGTTCCTGACCAAGGCG
T
D
D
316 CAGCCTGGTTCGTGCCCTGCACCCGCCACCAGTATACATGCTCTGGTCTA S
L
1716 CTAAACTGATGTAGCAACAGTTGTMTCAGMCCTMCMCTGCAAMTG 1766 TATCCTGTCAGCTTCTCMCTACTATGTGMGGACAAGTACACTCTGCGT
P
266 TCCMGGCGGCCAGCCGGGCGGCGCTCACCACCCGCTGCGCGATCGGCGA S
A
K
216 AGTCGCTGCGGGCGMCACGGCGAGGACGTCGTTCCCGGCGGGGCGGCAG S
V
1616 GMGATGTTCTCCCCTCGCMTCTGAGGGCCACGTTCCACMCCCTGATT
166 TGGGCAATGCGTGCGGCAGGCTGCCAGGCCGGCGACCTCGTTCTACGGCG A
L
K
116 TATCCAACATGTGGTCAGGCGTCAACCGTTTTTGTTTTGCTGATGCCGGA A
V
1516 T G G A G C T T C T G T A T G C G T G T G A C A G T G C T M T C ~ G G T C T A C T A C ~ T 1566 GCTGCAGGTAAATGGCAGCATGTCMG~CACCACCAGCATCGGAGMGG
A
66 CGCCATGTCCTTCTCGCAGTCCTACAGGCCCAAGGTGTGTMTGTTAGGA A
F
1466 C T C C T C G A C G M G G T T T T T A C T T C T C T T C T C A A T C M G T C C C C M T G G M
A
2766 2016 2066 2916 2966 3016 3066 31 16
L
A
A
S
A
T
V
G
A
T
A
*
TCTTCTTCTTTTTTTACGCCTTTCTCAACCTG~TTTAAACMGCTGT CCGTTATACAGTCTGMGCATGCTGTTGTCTTGTGATCATCC~CTGGT MTACAGTCGGCAGACCGGCTTGCGTACGTCTGCCGCCCTTGTCTCCACT GCGAGGCCATTGTTGTACGTACACAGTCGTGCGTTTGTGCATGGCATTGG
CMCACTTGTGTCATCACATGTCTGCGTGTCTMCCTMGCAGGCMCGT GATCTCCGATCTCTGCACCTTTGCTCCTCTCCCTCTCTCTCGCACACACA GGACTGTCACGCMCACTCCCGATGTTTCCACCAGCTACTACCAGCCAGG CCGCATGCMCAGTCCACGGTATGGGGT
Fig. 3. Nucleotide sequence of genomic clones coding for wheat Sed(l,7)P2ase. The deduced amino acid sequence is given below the nucleotide sequence. Nucleotides are numbered at the ends of lines, with the first letter of the initiating ATG codon as 1.
to suggest tentatively that the cleavage site of wheat Sed(l,7)P2ase may be in the region Arg59-Cys66, giving a processed subunit polypeptide of 328 - 334 amino acids, with a molecular mass of around 36 kDa, which is in agreement with previous data for maize (Nishizawa and Buchanan, 1981) and spinach (Cadet et al., 1987) Sed(l,7)P2ase. Comparison of Sed(l,7)P2ase and Fru(l,6)Pzase amino acid sequences
In order to determine the relationship between Sed(1,7)P2ase and Fru(1,6)P2ase, pairwise comparisons were
made between the derived amino acid sequence of wheat Sed(l.7)P2ase and Fru(l,6)P2ase from a variety of sources (Table 1) using the Wisconsin GAP programme (Devereux et al., 1984). This reveals that 29-32% of residues in Sed(1,7)P2aseare identical to those in all of the Fru(1 ,6)P2ases examined (including the plant cytosolic enzyme) and, if conservative changes are taken into account, this similarity rises to around 52%. Alignment of the wheat Sed(l,7)P2ase amino acid sequence with those of the wheat chloroplast and pig kidney Fru(l,6)P2asesis shown in Fig. 4. Although all three enzymes have identical residues in only 19% of the positions (42%
1057 Table 1. Identity and similarity between wheat Sed(l,7)Pzase and a variety of Fru(l,6)Pzase amino acid sequences. Sources of sequences: wheat Sed(1,7)P2ase, this work; wheat chloroplast Fru(l,6)P2ase, Raines et al. (1988); spinach cytosolic Fru(l,6)P2ase, Ladror et al. (1990); pig Fru(l,6)P2ase, Marcus et al. (1982); Saccharomyces cerevisiae Fru(l,6)P2ase, Rogers et al. (1988); E. coli Fru(l,6)P2ase, Hamilton et al. (1988); Rhodobacrer sphoeroides Fru(l,6)P2ase, Gibson et al. (1990). The alignments used to generate these values were made using the Wisconsin GAP programme.
Enzyme
Source
Identity with wheat Sed (1,7)P2ase
Fru( 1,6)P2ase from wheat chloroplast
spinach cytosol
pig
S. cerevisiae
E. coli
R. sphaero ides
%
Sed( I ,7)P2ase
wheat
-
29.6
29.8
31.8
28.1
31.8
30.0
Fru(l,6)P2ase
when chloroplast spinach cytosol Pig S . cerevisiae E. coli R . sphoreroides
51.5 52.7 53.7 52.0 53.3 53.0
-
50.0
69.5 63.1 65.7 64.6 59.0
-
44.7 51.8
73.9 71.1 62.8 52.0
-
41 .O 48.6 47.6
68.0 60.5 57.0
67.8 58.0
46.9 44.0 42.6 43.0 58.0
35.0 32.0 34.0 32.0 37.0
wfbp
1
-
40
20
WDTASAPAPAAARKRSSYDMITLTTWLLKQEQEGVIDNEMTIVLSSISTACKQIASLVQRA
PfbP
:: I : I::: :I :: I : 1 1 I: I 11 : : --------QSKAASRAALTTRCAIGDSLEEFLTKATPDKNLIRLLICMGEAMRTIAFKVRTA : I I :: : I :: 1 1 I( : )::I: 1 1 1 -----------TDQAAFDTNIVTLTRFVMEQGRKARGTGEMTQLLNSLCTAWISTAVRKA
wfbp
PISNLTGVQGATNVQGEDQXKLVISNEVFSNCLRWSGRTGVIASEEEDVPVAVEESYSGNY
wsbp
80
60
wsbp PfbP wfbp
I
1::l
I
:I::::
100
I :I
.. ..
Ill
I : I : I 1::I :1:1:: I: : 1 1 :I1 1 : GIAHLYGIAGSTNVTGDPVKKLDVLSNDLVINVLKSSFATCVLVTEEDXNAIIVEPEKRGKY S----
:
:: :
CGGTACVNSFGDEQLAVDMLADKLLFEALEYSHVCKYACSEEVPELQDMGGPVEGGF
+
120
**
140
160
wsbp
IWFDPLDGSSNIDAAVSTGSIFGIYSPSDECHIGDDATLDEVTQMCIVMlCQPGSNLLAAG I:: : I I : I I I I I I I I I :I : 1:lIl:: I II SVAFDPLDGSSIVDTNFTVGTIFGVWP--------- GDKLTGVT---------- GGDQVAAA
PfbP
WCFDPLDGSSNIDCLVSIGTIFGIYWOJSTDEPSEKDAL------------ QPERNLVAAG
I I I I I I I I I :I 4
::l:lll::
I I
Ill:
:
*+
200
180
220
wfbp
YCMYSSSVIFVLTIGTGVYVFTLDPMYGEFVLTQEKQIPKSGKIYSFNEGNYALWDDKL-K
wsbp
MGIYGPRTTFWALKDCPGTHEFLLLDEGKWQHVKDTTSIGTFDNP-:I:: I :I::: : I ::I I II
PfbP
.. ..
:I:::
:I::
.. ..
ll::l
I
I1
YALYGSA~LVLAMVNGVNC~DPAIGEFILVDRNVKIKKKGSIYSINEG-YAKEFDPAIT
+
240
260
+
280
wf bp
XYMDSLKEPGTSGKPYSARYIGSLVGDFHRTMLYG-GIYGYPSDQKSKNGKLRUYECAPMS
wsbp
DYDKLVNY~EK--YTLRYTGGMVPDVNQIIVKEKGIFTLRLLFEVAPLG
PfbP wfbp wsbp pfbp
-
I : ) I I::I I :: :: 1 1 : : : : : ~11::~ :: : 1 : 1 1 I : I ) 1 1 : : :: 1 1 1 : : I:I1III:I I:: EYIERKXFPPDNSAPYGARYVGSMVADVHRTLVYG-GIFMYPANKKSPKGKLRLLYECNPMA ** *+* + + * * I :I
::
~
~
~
:
300 320 FIAEQAGGKGSDGHQRVLDIlrIPTAVHQRVPLYVGSVEEEKFLSSE-----------I: I : J I I 1 : : :::I : :I: 1 : :I FLIEKAGGHSSDGKQSVLDKTSVLDERTQVAYGSKNEIISATVGATA : : : ::~ ~ I ~ I : ~ ~: 1 1 ::: : 1
:Ill
Il::ll
111
11
:::I:
YVMEKAGGLATTGKEAVLDIVPTDIHQRAPIILGSPEDVTELLEIYQKHA------------
Fig. 4. Amino acid sequence alignment of wheat chloroplast Sed(l,7)PZase(wsbp) with wheat chloroplast Fru(l,6)Pzase (wfbp; Raines et al., 1988) and pig kidney Fru(l,6)Pzase (pfbp; Marcus et al., 1982). The amino acids are numbered according to the pig kidney sequence and the transit peptides of the chloroplast enzymes are not shown. Identical residues are indicated by solid lines and conservative changes with a dotted lines. * or denotes residues implicated in Fru(l,6)P2ase substrate binding (Ke et al., 1989b) which are either present ( * ) or absent (+) in Sed(l,7)P2ase.
+
including conservative changes), it is obvious from this alignment that, rather than being evenly dispersed throughout the sequence, there are distinct regions where the sequence is
highly conserved. These regions correspond to blocks of identity which have been found to exist in all Fru(l,6)P2ases studied to date, and contain the residues which have been
~
1058 Table 2. Comparisonof amino acid residues in Fru(l,6)Pzaseimplicated in substrate binding with residues at identical positions in wheat Sed(l,7)P2ase. + indicates the presence of the same residue as that found in the pig sequence. - indicates that a gap was introduced into the alignment at the corresponding position. The figures in brackets are the N B R F point acceptable values. Non-conservative changes score negative values and conscrvative changes either zero or positive values. ~~
Substrate part
Fru(l,6)P2ase from Pig
Wheat Sed( 1,7)P2ase
wheat ~
6-phosphate
Sugar group
( (2)-phosphate
Arg243 Tyr244 Am212 Tyr215 Tyr264 Lys269 Lys274 Glu280 Arg276 Gly246 Ser241 Met248 Asp121 Lys274 Lys274 Arg216 Ser124
+ + + + + + + + + + + Leu (4) + + + + +
+ + Leu ( -2) Asn (-4) -
+ + + + GlY (1) + + + + + +
implicated in binding of the substrate analogue Fru(2,6)P2 (Ke et al., 1989a, b) and the reaction product fructose 6-phosphate (Fru6P; Ke et al., 1990, 1991a, b). Active site residues The overall degree of similarity between the bisphosphatases is sufficient to allow us to make use of the structural information available for pig kidney Fru( 1 ,6)P2ase. The location of the active site was determined from crystallographic studies using the substrate analogue /?-~-Fru(2,6)P,(Ke et al., 1989a, b) and with the product Fru6P (Ke et al., 1990,1991 a, b). If we consider the 19 residues implicated in substrate binding to pig kidney Fru(l,6)P2ase (Ke et al., 1989b) then Sed(1,7)P2ase contains 12 of these residues. The information presented in Table 2 shows that the amino acids which would bind to the sugar moeity are highly conserved, as are the residues which would interact with the phosphate at C1 which is removed by the enzyme. However, the residues which interact with the stable phosphate at C6 in pig and wheat Fru(l,6)P2ases are not so well conserved in Sed(1,7)P2ase and may account for the different substrate specificities of the two enzymes. The residues Aspll8, Asp121 and Glu280, which bind Mg2+ close to the active site in Fru(l,6)P2ase, are all conserved in Sed(l,7)P2ase. Sed(l,7)Plase activation In vivo, the activities of Sed(l,7)P2ase (Breazeale et al., 1978; Wirtz et al., 1982; Cadet and Meunier, 1988a) and chloroplast Fru(1 ,6)P2ase (Zimmerman et al., 1976) are modulated by reduced thioredoxin f which is generated by the light reactions of photosynthesis. The wheat and spinach chloroplast Fru(1 ,6)P2ases have been shown to have a unique insertion sequence (at position
Leu1 53 in the pig sequence), containing two cysteine residues which may be involved in the light regulation of these enzymes (Raines et al., 1988; Marcus et al., 1988). The alignment in Fig. 4 reveals that the wheat chloroplast Sed(1,7)P2ase contains no such insertion and, indeed, none of the cysteines present in Fru(1 ,6)P2ase and Sed(1,7)P2ase align with one another, suggesting that the light-activation region in these two enzymes is different, despite their overall similarities. Sed(t ,7)P2ase has seven cysteines, comprising two pairs and three single residues. One pair of cysteines is separated by four intervening residues (CGGTAC), a structural arrangement which is found for the redox-active groups of a number of enzymes, including mercuric reductase, glutathione reductase, lipoamide dehydrogenase (Fox and Walsh, 1983) and two ribonucleotide reductases (Lin et al., 1987). A subset of these, whch includes Fru(1 ,6)P2ase but not Sed(l,7)P2ase, also share greater similarity of the residues flanked by the cysteine pair (Marcus et al., 1988).
DISUSSION Sed(l,7)Pzase is unique to photosynthetic organisms, where it functions in the photosynthetic carbon-reduction cycle. This enzyme shares a number of functional similarities with the chloroplast Fru(l,6)P2ase, therefore one might expect these enzymes to have structural features in common. However, to date there is very little information on the molecular characteristics of Sed(1,7)P,ase, and both immunological studies and amino acid composition data have emphasised the differences between Fru(1 ,6)P2ase and Sed(1,7)P2ase (Cadet and Meunier, 1988a). Our sequence comparisons (Fig. 4) have shown that there is appreciable similarity between the wheat Sed(1,7)P2ase and Fru(1 ,6)P2ases from diverse sources. The most interesting aspect of our analysis is that the active-site residues shown by crystallographic analysis of pig kidney Fru(l,6)P2ase (Ke et al., 1989a, b, 1990, 1991a, b) are conserved in Sed(1,7)P2ase. Our results support biochemical data which indicate that the chloroplast Sed(1,7)P2ase molecule has one active site which is capable of binding either Sed(1,7)Pz or Fm(l ,6)P2 (Cadet and Meunier, 1988b). These authors also suggested that negative charges would be present near the active site of Sed(1,7)P2ase. The alignment in Fig. 4 shows that all the residues which form a negatively charged pocket, comprising one wall of the binding site of Fru(l,6)P2ase (Glu97 and Glu98, Asp118 and Aspl21, Glu280), are also found in wheat Sed(l,7)P2ase. With the availability of the wheat cDNA clone encoding this enzyme it will now be possible to test these hypotheses using a combination of in vitro mutagenesis and expression of the protein in Esckerichia coli. The similarity which exists between Sed(l,7)P2ase and chloroplast Fru(1,6)P2ase raises the question of the mechanism by which these enzymes evolved. Sed(l,7)P2ase might have arisen from a common bisphosphatase progenitor before the development of oxygenic photosynthesis, or diverged more recently from a bifunctional Sed(1,7)P2ase/Fru(l,6)P2ase enzyme such as that found in cyanobacteria (Gerbling et al., 1986). Although unlikely, convergent evolution of these enzymes cannot be ruled out. More information will be needed on the occurrence and properties of Sed(l,7)Pzase in prokaryotes to indicate which of these explanations is more likely. We have compared the structure of the wheat Sed(1,7)P2ase gene (seven introns) with those available for Fru(l,6)P2ase, namely the wheat chloroplast (three introns;
1059 Lloyd et al., 1991) and rat liver genes (six introns; ElMaghrdbi et al., 1991). No introns are found at identical positions in these genes, although intron 111 in the wheat Sed(l,7)P2ase gene is within four codons of intron I of wheat chloroplast Fru(l,6)P2ase, and introns IV and V of are one and four codons away from the positions of introns I1 and IV of rat liver Fru(l,6)P2ase, respectively. It seems unlikely that these observations are of any evolutionary significance, as the two Fru(1 ,6)P2ase genes themselves show least correspondence of intron positions. Evidence from biochemical studies, including control analysis, has shown that the activity of Sed(1,7)P2aseis tightly regulated. which has led to the belief that this enzyme plays a key role in controlling the flow of intermediates through the photosynthetic carbon reduction cycle (Woodrow, 1986; Woodrow and Berry, 1988; Pettersson and Ryde-Pettersson, 1989). The isolation of sequences encoding wheat Sed(1,7)P2aseshould now allow this to be tested using transgenic plants with altered amounts of this enzyme. We would like to thank Professor B. B. Buchanan for the Sed(1 ,7)P2ase antibodies. This work was supported by the Royal Society (J.C.L.). an AFRC PMB grant (S.P.) and a SERC studentship (N.M.W.). C. A. Raines and J. C. Lloyd both made equal contributions to this work.
REFERENCES Baulcombe. D.C. & Buffard, D. (1983) Planta (Berl.) 157,493-501. Breazeale, V. D.. Buchanan, B. B. & Wolosiuk, R.A. (1978) Z. Naturforsch. 33c, 521 - 528. Cadet, F.. Meunier, J.-C. & Ferte. N. (1987) Biochem. J . 241, 71 -74. Cadet, F. & Meunier. J.-C. (1988a) Biochem. J. 253,243-248. Cadet. F. & Mcunier, J.-C. (1988b) Biochem. J. 253, 249-254. Devereux. J.. Haeberli, P. & Smithies, 0. (1984) Nucleic Acids Res. 12,387-395. El-Maghrabi, M. R., Lange, A. J., Kummel, L. & Pilkis, S. J. (1991) J . Biol. Cliem. 266. 2115-2120. Fcinberg, A.P. & Vogelstein. B. (1983) Anal. Biochem. 132, 6-13. Fox. B. S. & Walsh. C. T. (1983) Biuchemistry 22,4082-4088. Gerbling. K.-P.. Steup, M. & Latzko, E. (1986) Plant Physiol. 80, 716-720. Gibson. J. L., Chen, J. H., Tower, P. A. & Tabita, F. R. (1990) Biochemistry 2Y, 8085 - 8093. Hamilton. W. D. 0.. Harrison, D. A. & Dyer, T. A. (1988) Nucleic Acids Rex 16. 8707. Henikoff. S . (1984) Gene (Amst.) 28, 351 -359. Huttly, A . K., Martienssen, R. A. & Baulcombe, D. C. (1988) Mol. Gen. Genet. 214, 232 - 240. Ke, H.. Thorpe. C. M., Seaton. B. A., Marcus, F. & Lipscomb, W. N. (1989a) Proc. Natl Acad. Sri. U S A 86, 1475-1479.
Ke. H., Thorpe. C. M., Seaton, B. A., Lipscomb, W. N. & Marcus, F. (1989b) J. Mol. Biol. 212, 513-539.Ke, H., Zhang, Y. & Lipscomb, W. N. (1990) Pruc. Natl Acud Sci. USA 87, 52435247. Ke, H., Zhang, Y.. Liang, J.-Y. & Lipscomb, W.N. (19Yla) Proc. Natl Acud Sci. USA 88, 2989 - 2993. Ke. H., Liang, J.-Y., Zhang, Y. & Lipscomb. W.N. (1991 b) Biuchemistry 30,441 2 - 4420. Ladror, U. S., Latshaw, S. P. & Marcus. F. (1990) Eur. J . Biochem. 189, 89 - 94. Lin, A.-E. I. , Ashley, G. W. & Stubbe, J. (1987) Biochemistry 26, 6905 - 6909. Lloyd, J. C., Raines, C. A., John, U. P. & Dyer, T. A. (1991) Mol. Gen. Genet. 225. 209 - 216. Longstaff, M., Raines, C. A,, McMorrow, E. M., Bradbeer, J . W. & Dyer, T. A. (1989) Nucleic Acids Res. 17, 6569-6580. Marcus, F., Edelstein, I., Reardon, I. & Heinrikson, R. L. (1982) Proc. Natl Acad. Sci. USA 79, 7161 -7165. Marcus, F., Moberly, L. & Latshaw, S. P. (1988) Proc. Natl Acad. Sci. U S A 85, 5379 - 5383. Murphy, G. J. P. & Ward, E. S. (1990) in Nucleic acids sequencing a practical approach (Howe, C. J. & Ward, E. S. eds) pp. 99 115, IRL Press, Oxford. Nishizawa, A. N. & Buchanan, B. B. (1981) J . Biol.Cheni. 256,61196126. Petterson, G. & Ryde-Petterson, U. (1989) Eur. J . Biuchem. 186,683 687. Raines, C. A,, Lloyd, J. C., Longstaff, M., Bradley, D. & Dyer, T. A. (1988) Nucleic acid^ Res. 16, 7931 -7942. Rogers, D. T., Hiller, E., Mitsock. L. & Orr. E. (1988) J. B i d . Chem. 263,6051 -6057. Sambrook, J.. Fritsch, E. F. & Maniatis, T. (1989) Molecular cloning: a Iuboratory manual, 2nd edn, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Schimkat, D., Heincke, D. & Heldt, H. W. (1990) Planta (Berl.) 181, 97-103. Wirtz, W., Stitt, . & Heldt, H. W. (1982) FEBS Lett. 142, 223-226. Woodrow, I. E. &Walker, D. A. (1Y82) Arch. Biochem. Biophys. 216, 416 -422. Woodrow, 1. E.. Murphy, D. J. & Walker, D. A. (1983) Eur. J . Biochem. 132, 121 - 123. Woodrow, I. E., Murphy, D. J. & Latzko. E. (1984) J . Biol. Chem. 259, 3791 -3795. Woodrow, I. E. & Berry. J. A. (1988) Annu. Rev. Plant Physiol. Plunt Mol. Biol. 39, 533 - 594. Woodrow, T. E. (1986) Biochim. Biophys. Acta 851, 181 - 192. Von Heijne, G., Steppuhn, J. & Hermann, R. G. (1989) Eur. J . Biochem. 180, 535 - 545. Young, R. A. & Davis, K. W. (1983) Science 222, 778-782. Zimmermann, G.. Kelly, G. J. & Latzko, E. (1976) Eur. J. Biochem. 70, 361 - 367.
