Abstract
A cDNA (VUpur5) encoding phosphoribosyl aminoimidazole (AIR) synthetase, the fifth enzyme of the de novo purine biosynthesis pathway has been isolated from a cowpea nodule cDNA library. It encodes a 388 amino acid protein with a predicted molecular mass of 40.4 kDa. The deduced amino acid sequence has significant homology with AIR synthetase from other organisms. AIR synthetase is present in both mitochondria and plastids of cowpea nodules [7]. A signal sequence encoded by the VUpur5 cDNA has properties associated with plastid transit sequences but there is no consensus cleavage site as would be expected for a plastid targeted protein. Although the signal sequence does not have the structural features of a mitochondrial targeted protein, it has a mitochondrial cleavage site motif (RX/XS) close to the predicted N-terminus of the mature protein. Southern analysis suggests that AIR synthetase is encoded by a single gene raising questions as to how the product of this gene is targeted to the two organelles. VUpur5 is expressed at much higher levels in nodules compared to other cowpea tissues and the gene is active before nitrogen fixation begins. These results suggest that products of nitrogen fixation do not play a role in the initial induction of gene expression. VUpur5 was expressed in Escherichia coli and the recombinant protein used to raise antibodies. These antibodies recognize two forms of AIR synthetase which differ in molecular size. Both forms are present in mitochondria, although the larger protein is more abundant. Only the smaller protein was detected in plastids.
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Atkins CA, Pate JS, Griffith GJ, White ST: Economy of carbon and nitrogen in nodulated and nonnodulated (NO3 grown) cowpea (Vigna unguiculata (L.)Walp.) Plant Physiol 66: 978–983 (1980).
Atkins CA, Ritchie A, Rowe PB, McCairns E, Sauer D: De novo purine synthesis in nitrogenfixing nodules of cowpea (Vigna unguiculata L. Walp) and soybean (Glycine max L. Merr). Plant Physiol 70: 55–60 (1982).
Atkins CA, Pate JS, Shelp BJ: Effects of shortterm N2 deficiency on N metabolism in legume nodules. Plant Physiol 76: 705–710 (1984).
Atkins CA, Sanford P, Storer PJ, Pate JS: Inhibition of nodule functioning in cowpea by a xanthine oxidoreductase inhibitor, allopurinol. Plant Physiol 88: 1229–1234 (1988).
Atkins CA: Ammonia assimilation and export of nitrogen from the legume nodule. In: Dilworth MJ, Glenn AR (eds) Biology and Biochemistry of Nitrogen Fixation, pp. 293–319, Elsevier Science Publishers, Amsterdam (1991).
Atkins CA, Fernando M, Hunt S, Layzell DT: A metabolic connection between nitrogenase activity and the synthesis of ureides in nodulated soybean. Physiol Plant 84: 441–447 (1992).
Atkins CA, Smith PMC, Storer PJ: Reexamination of the intracellular localization of de novo purine biosynthesis in cowpea nodules. Plant Physiol 113: 127–135 (1997).
Boland MJ, Schubert KR: Biosynthesis of purines by a proplastid fraction from soybean nodules. Arch Biochem Biophys 220: 179–187 (1983).
Chapman KA, Delauney AJ, Kim JH, Verma DPS: Structural organization of de novo purine biosynthesis enzymes in plants-5-aminoimidazole carboxamide ribonucleotide carboxylase and 5-aminoimidazole-4-N-succino-carboxamide ribonucleotide synthetase cDNA's from Vigna aconitifolia. Plant Mol Biol 24: 389–395 (1994).
Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ: Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294–5299 (1979).
Chomczynski P, Sacchi N: Singlestep method of RNA isolation by acid guanidinium thiocynatephenolchloroform extraction. Anal Biochem 162: 156–159 (1987).
Chung KH, Korban SS, Buetow DE: Construction of a genomic library using highmolecularweight DNA prepared from peach leaves. Plant Mol Biol Rep 12: 304–309 (1994).
Claros MG: MitoProt, a Mackintosh application for studying mitochondrial proteins. CABIOS 11: 441–447 (1995).
D' Alessio JM, Bebee R, Hartley JL, Noon MC, Polayes D: Lambda ZIPLOXTM: automatic subcloning of cDNA. Focus 14: 76–79 (1992).
Devereaux J, Haeberli P, Smithies O: A comprehensive set of sequence analysis programs for the VAX. Nucl Acids Res 12: 387–395 (1984).
Gavalas A, Zalkin, H: Analysis of the chicken GPAT/AIRC bidirectional promoter for de novo purine nucleotide synthesis. J Biol Chem 270: 2402–2410 (1995).
Gavel Y, von Heijne G: Cleavagesite motifs in mitochondrial targeting peptides. Prot Engin 4: 33–37 (1990).
Gavel Y, von Heijne G: A conserved cleavagesite motif in chloroplast targeting peptides. FEBS Lett 261: 455–458 (1990).
Hall DJ, Atkins CA: Purification of glycinamide ribonucleotide transformylase from root nodules of cowpea (Vigna unguiculata). Use of the novel affinity resin 5,10dideaza5,6,7,8tetrahydrofolate sepharose. In Randall DD, Sharp RE, Novacky AJ, Blevins DG(eds) Current Topics in Plant Biochemistry and Physiology, vol 11, p. 302. University of MissouriColumbia, Columbia (1992).
He B, Shiau A, Choi KY, Zalkin H, Smith JM: Genes of the Escherichia coli pur regulon are negatively controlled by a repressoroperator interaction. J Bact 172: 4555–4562 (1990).
He B, Zalkin H: Regulation of Escherichia coli purA by purine repressor, one component of a dual control mechanism. J Bact 176: 1009–1013 (1994).
Kim JH, Delauney AJ, Verma DPS: Control of de novo purine biosynthesis genes in ureideproducing legumesInduction of glutamine phosphoribosylpyrophosphate amidotransferase gene and characterization of its cDNAfrom soybean and Vigna. Plant J 7: 77–86 (1995).
Laemmli UK: Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227: 680–685 (1970).
Lee NG, Stein B, Suzuki H, Verma DPS: Antisense expression of nodulin-35 RNA in Vigna aconitifolia root nodules retards peroxisome development and the availability of nitrogen to the plant. Plant J 3: 599–606 (1993).
McGadey J: A tetrazolium method for nonspecific alkaline phosphatase. Histochemie 23: 180–184 (1970).
Meng LM, Kilstrup M, Nygaard P: Autoregulation of PurR repressor synthesis and involvement of PurR in the regulation of purB, purC, purL, purMN, and guaBA expression in Escherichia coli. Eur J Biochem 187: 373–379 (1990).
Rolfes RJ, Zalkin H: Purification of the Escherichia coli purine regulon repressor and identification of corepressors. J Bact 172: 5637–5642 (1990).
Ross CW: In: Stumph PK, Conn EE (eds) The Biochemistry of Plants, vol. 6, pp. 169–205. Academic Press, New York (1981).
Rothnie HM: Plant mRNA 3′end formation. Plant Mol Biol 32: 43–61 (1996).
Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).
Sanger F, Nicklen S, Coulson AR: DNAsequencing with chain terminatinginhibitors. Proc Natl Acad Sci USA 74: 5463–5467 (1977).
Schrimsher JL, Schendel FJ, Stubbe J: Isolation of amultifunctional protein with aminoimidazole ribonucleotide synthetase, glycinamide ribonucleotide synthetase, and glycinamide ribonucleotide transformylase activities: Characterization of aminoimidazole ribonucleotide synthetase. Biochemistry 25: 4356–4365 (1986).
Schrimsher JL, Schendel FJ, Stubbe J, Smith JM: Purification and characterization of aminoimidazole ribonucleotide synthetase from Escherichia coli. Biochemistry 25: 4366–4371 (1986).
Schnorr KM, Laloue M, Hirel B: Isolation of cDNAs encoding two purine biosynthetic enzymes of soybean and expression of the corresponding transcripts in roots and root nodules. Plant Mol Biol 32: 751–757 (1996).
Senecoff J, Meagher R: Isolating the Arabidopsis thaliana genes by suppression ofEscherichia colimutants. Plant Physiol 102: 387–399 (1993).
Shelp BJ, Atkins CA, Storer PJ, Canvin DT: Cellular and subcellular organization of pathways of ammonia assimilation and ureide synthesis in nodules of cowpea (Vigna unguiculata L. Walp). Arch Biochem Biophys 224: 429–441 (1983).
Smith JM, Duam HA: Nucleotide sequence of a gene encoding 5′phosphoribosyl-5-aminoimidazole synthetase of Escherichia coli K12. J Biol Chem 261: 10632–10636 (1987).
Towbin H, Staehlin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354 (1979).
Von Heijne G, Steppuhn J, Hermann RG: Domain structure of mitochondrial and chloroplast targeting peptides. Eur J Biochem 180: 535–545 (1989).
Winning BM, Sarah CJ, Purdue PE, Day CD, Leaver CJ: The adenine nucleotide translocator of higher plants is synthesized as a large precursor that is processed upon import into mitochondria. Plant J 2: 763–777 (1992).
Wu L, Ueda T, Messing J: The formation of mRNA 3′ends in plants. Plant J 8: 323–329 (1995).
Zalkin H, Dixon JE: De novo purine nucleotide biosynthesis. Prog Nucl Acids Res Mol Biol 42: 259–287 (1992).
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Smith, P.M., Mann, A.J., Goggin, D.E. et al. Air synthetase in cowpea nodules: a single gene product targeted to two organelles?. Plant Mol Biol 36, 811–820 (1998). https://doi.org/10.1023/A:1005969830314
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DOI: https://doi.org/10.1023/A:1005969830314