Proc Natl Acad Sci U S A 97(15): 8705-8710

Adenosine diphosphate glucose pyrophosphatase: A plastidial phosphodiesterase that prevents starch biosynthesis

Instituto de Agrobiotecnología y Recursos Naturales, Universidad Pública de Navarra /Consejo Superior de Investigaciones Científicas, Carretera de Mutilva s/n, Mutilva Baja, 31192 Navarra, Spain

^{To whom reprint requests should addressed. E-mail:}se.arravanu@ateuzop.reivaj.

Communicated by André T. Jagendorf, Cornell University, Ithaca, NY

Received 1999 Nov 28; Accepted 2000 Apr 13.

Abstract

A distinct phosphodiesterasic activity (EC 3.1.4) was found in both mono- and dicotyledonous plants that catalyzes the hydrolytic breakdown of ADPglucose (ADPG) to produce equimolar amounts of glucose-1-phosphate and AMP. The enzyme responsible for this activity, referred to as ADPG pyrophosphatase (AGPPase), was purified over 1,100-fold from barley leaves and subjected to biochemical characterization. The calculated K_eq′ (modified equilibrium constant) value for the ADPG hydrolytic reaction at pH 7.0 and 25°C is 110, and its standard-state free-energy change value (ΔG′) is −2.9 kcal/mol (1 kcal = 4.18 kJ). Kinetic analyses showed that, although AGPPase can hydrolyze several low-molecular weight phosphodiester bond-containing compounds, ADPG proved to be the best substrate (K_m = 0.5 mM). P_i and phosphorylated compounds such as 3-phosphoglycerate, PP_i, ATP, ADP, NADP^{, and AMP are inhibitors of AGPPase. Subcellular localization studies revealed that AGPPase is localized exclusively in the plastidial compartment of cultured cells of sycamore (}Acer pseudoplatanus L.), whereas it occurs both inside and outside the plastid in barley endosperm. In this paper, evidence is presented that shows that AGPPase, whose activity declines concomitantly with the accumulation of starch during development of sink organs, competes with starch synthase (ADPG:1,4-α-d-glucan 4-α-d-glucosyltransferase; EC 2.4.1.21) for ADPG, thus markedly blocking the starch biosynthesis.

Abstract

Although the pyrophosphorolytic reactions leading to the production of gluconeogenic intermediates such as ADPglucose (ADPG) and UDPglucose (UDPG) are readily reversible, they mainly proceed toward the direction of nucleotide sugar synthesis (1). On the other hand, plant enzymes that irreversibly cleave nucleotide sugars have been described that may effectively interrupt the flow of glycosyl moieties toward the biosynthesis of end products such as starch, cell wall polysaccharides, or sucrose (2). It is conceivable that in conjunction with ADPG pyrophosphorylase (AGPase; EC 2.7.7.27), UDPG pyrophosphorylase, sucrose synthase, and starch synthase, among others, these enzymes will participate in controlling the levels of nucleotide sugars engaged in starch formation. Among a few enzymes reported to date that can hydrolyze nucleotide sugars in plants, ADPG phosphorylase is shown to catalyze the phosphorolytic breakdown of ADPG (3). UDPG phosphorylase is known to split UDPG in the presence of P_i, and its activities are greatly stimulated by some signal metabolites, indicating that it may play an important role in the control of photosynthate partitioning (4).

Based on experimental grounds, Pozueta-Romero et al. (5) have proposed the operation of synthesis/breakdown metabolic cycles controlling the rate of starch formation. According to this hypothesis, the balance between enzymatic activities catalyzing the synthesis of gluconeogenic intermediates and those activities catalyzing their breakdown can determine the net rate of starch synthesis. Because of the possibility that activities hydrolyzing ADPG, the universal starch precursor, may exist in plants that regulate the metabolism of this polyglucan, we have explored their possible occurrence in several plant species. As a result, we have now found a phosphodiesterasic activity that catalyzes the hydrolytic breakdown of ADPG. In this paper, we report the subcellular localization and biochemical characterization of the enzyme responsible for this activity, referred to as ADPG pyrophosphatase (AGPPase).^† Based on the results presented in this work using different plant sources, we discuss that AGPPase may be involved in controlling the intracellular levels of ADPG linked to starch biosynthesis.

Data are presented as the mean ± SD obtained from five independent experiments. Details of enzyme assay methods to measure G1P production are described in Material and Methods.

Kinetic parameters (K_m, V_max, K_i, and stoichiometry) obtained are the mean values from five independent experiments. NQ, not quantifiable.

Kinetic parameters (K_m, V_max, K_i, and stoichiometry) obtained are the mean values from five independent experiments. ND, not detectable.

Data are presented as the mean ± SD obtained from six separate preparations.

Abbreviations

ΔG′	standard-state free-energy change
ADPG	ADPglucose
AGPase	ADPG pyrophosphorylase
AGPPase	ADPG pyrophosphatase
G1P	glucose-1-phosphate
3-PGA	3-phosphoglycerate
PNPP	p-nitrophenyl phosphate
UDPG	UDPglucose

Abbreviations

Footnotes

^{AGPPase also can be referred to as adenosine diphosphate glucose phosphodiesterase.}

Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.120168097.

Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.120168097

Footnotes

References

1. Preiss J In: The Biochemistry of Plants. Preiss J, editor. Vol. 14. New York: Academic; 1988. pp. 181–254. [PubMed][Google Scholar]
2. Feingold D S, Avigad G In: The Biochemistry of Plants. Stumpf P K, Conn E E, editors. Vol. 3. New York: Academic; 1980. pp. 101–170. [PubMed][Google Scholar]
3. Murata T. Agric Biol Chem. 1977;41:1995–2002.[PubMed]
4. Gibson D M, Shine W. Proc Natl Acad Sci USA. 1983;80:2491–2494.
5. Pozueta-Romero J, Perata P, Akazawa T. Crit Rev Plant Sci. 1999;18:489–525.[PubMed]
6. Pozueta-Romero J, Frehner M, Viale A M, Akazawa T. Proc Natl Acad Sci USA. 1991;88:5769–5773.
7. Thorbjornsen T, Villand P, Denyer K, Olsen O-A, Smith A M. Plant J. 1996;10:243–250.[PubMed]
8. Reisfeld R A, Lewis U J, Williams D E. Nature (London) 1962;195:281–283.[PubMed]
9. Sowokinos J R. Plant Physiol. 1981;68:924–929.
10. Nishimura M, Beevers H. Plant Physiol. 1978;62:44–48.
11. Shannon J C, Pien F-M, Liu K-C. Plant Physiol. 1996;110:835–843.
12. Klotz I M Energy Changes in Biochemical Reactions. New York: Academic; 1967. [PubMed][Google Scholar]
13. Hill L M, Smith A M. Planta. 1991;185:91–96.[PubMed]
14. Smith A M, Bettey M, Bedford I D. Plant Physiol. 1989;89:1279–1284.
15. Lin T-P, Caspar T, Somerville C R, Preiss J. Plant Physiol. 1988;86:1131–1135.
16. Schliselfeld L H, van Eys J, Touster O. J Biol Chem. 1965;240:811–817.[PubMed]
17. Gasmi L, Cartwright J L, McLennan A G. Biochem J. 1999;344:331–337.
18. Melo A, Glaser L. Biochem Biophys Res Commun. 1966;22:524–531.[PubMed]
19. Sheikh S, O'Handley S F, Dunn C A, Bessaman M. J Biol Chem. 1998;273:20924–20928.[PubMed]
20. Cabib E, Carminatti H. J Biol Chem. 1961;236:883–887.[PubMed]
21. Bessman M J, Frick D N, O'Handley S F. J Biol Chem. 1996;271:25059–25062.[PubMed]
22. Gangwani L, Khurana J P, Maheshwari S C. Phytochemistry. 1994;35:857–861.[PubMed]
23. Harvey C L, Olson K C, Wright R. Biochemistry. 1971;9:921–925.[PubMed]
24. Lerch B, Wolf G. Biochim Biophys Acta. 1972;258:206–218.[PubMed]
25. Ito K, Yamamoto T, Minamiura N. J Biochem. 1987;102:359–367.[PubMed]
26. Culver G M, Consaul S A, Tycowski K T, Filipowicz W, Phizicky E M. J Biol Chem. 1994;269:24928–24934.[PubMed]
27. Preiss J, Shen L, Greenberg E, Gentner N. Biochemistry. 1966;5:1833–1845.[PubMed]
28. Shinshi H, Miwa M, Kato K, Noguchi M, Matsushima T, Sugimura T. Biochemistry. 1976;15:2185–2190.[PubMed]
29. Akazawa T. Plant Mol Biol Rep. 1991;9:145–155.[PubMed]
30. ap Rees T In: The Biochemistry of Plants. Stumpf P K, Conn E E, editors. Vol. 3. New York: Academic; 1980. pp. 1–42. [PubMed][Google Scholar]
31. Denyer K, Dunlap F, Thorbjornsen T, Keeling P, Smith A M. Plant Physiol. 1996;112:779–785.
32. Geigenberger P, Stitt M. Planta. 1991;185:81–90.[PubMed]
33. Preiss J. Oxford Surv Plant Mol Cell Biol. 1991;7:59–114.[PubMed]
34. Randall D D, Tolbert N E. Plant Physiol. 1971;48:488–492.
35. Pozueta-Romero J, Akazawa T. J Exp Bot. 1993;44,Suppl.:297–306.[PubMed]
36. Werdan K, Heldt H W, Milovancev M. Biochim Biophys Acta. 1975;396:276–292.[PubMed]
37. Sowokinos J R. Plant Physiol. 1976;57:63–68.
38. Prioul J-L, Jeannette E, Reyss A, Grégory N, Giroux M, Hannah L C, Causse M. Plant Physiol. 1994;104:179–187.
39. Schaffer A A, Petreikov M. Plant Physiol. 1997;113:739–746.
40. ap Rees T, Hill S A. Plant Cell Environ. 1994;17:587–589.[PubMed]
41. Stitt M, Sonnewald U. Annu Rev Plant Physiol Plant Mol Biol. 1995;46:341–368.[PubMed]
42. Murata T, Sugiyama T, Minamikawa T, Akazawa T. Arch Biochem Biophys. 1966;113:34–44.[PubMed]
43. Geigenberger P, Geiger M, Stitt M. Plant Physiol. 1998;117:1307–1316.
44. Geigenberger P, Müller-Röber B, Stitt M. Planta. 1999;209:338–345.[PubMed]