Identification of the amino acid involved in the regulation of bacterial pyruvate, orthophosphate dikinase and phosphoenolpyruvate synthetase
Affiliation(s)
Biochemistry and Molecular Biology, James Cook University, Townsville, Australia. Department of Biology, Minnesota State University, Moorhead, USA.
Biochemistry and Molecular Biology, James Cook University, Townsville, Australia. Department of Biology, Minnesota State University, Moorhead, USA.
ABSTRACT
Pyruvate, orthophosphate dikinase (PPDK) and phosphoenolpyruvate synthetase (PEPS) catalyze the conversion of pyruvate to phosphoenolpyruvate (PEP). Both are regulated by a phosphorylation-dephosphorylation mechanism involving a bifunctional serine/ threonine kinase and a pyrophosphorylase (PPDK regulatory protein, PDRP, and PEPS regulatory protein, PSRP, respectively). In plants the regulatory mechanism involves phosphorylation of a threonine residue that is separated by a single amino acid from the histidine residue that forms a phosphorylated intermediate during catalysis. Using antibodies, we demonstrated that the regulation of both Listeria monocytogenes PPDK and Escherichia coli PEP synthetase involves the phosphorylation of a threonine residue located close to the catalytic histidine residue. The amino acid located between the regulatory threonine and the catalytic histidine is highly conserved being serine in PPDK and cysteine in PEPS. Using site-directed mutagenesis we have shown that both PPDK and PEPS in which the serine and cysteine residues, respectively, were substituted with an alanine the enzymes could be regulated indicating that the serine and cysteine residues, respectively, are not essential for regulation. We also demonstrated that altering the intermediate amino acid did not alter the specificity of the regulatory proteins for their protein substrates.
Pyruvate, orthophosphate dikinase (PPDK) and phosphoenolpyruvate synthetase (PEPS) catalyze the conversion of pyruvate to phosphoenolpyruvate (PEP). Both are regulated by a phosphorylation-dephosphorylation mechanism involving a bifunctional serine/ threonine kinase and a pyrophosphorylase (PPDK regulatory protein, PDRP, and PEPS regulatory protein, PSRP, respectively). In plants the regulatory mechanism involves phosphorylation of a threonine residue that is separated by a single amino acid from the histidine residue that forms a phosphorylated intermediate during catalysis. Using antibodies, we demonstrated that the regulation of both Listeria monocytogenes PPDK and Escherichia coli PEP synthetase involves the phosphorylation of a threonine residue located close to the catalytic histidine residue. The amino acid located between the regulatory threonine and the catalytic histidine is highly conserved being serine in PPDK and cysteine in PEPS. Using site-directed mutagenesis we have shown that both PPDK and PEPS in which the serine and cysteine residues, respectively, were substituted with an alanine the enzymes could be regulated indicating that the serine and cysteine residues, respectively, are not essential for regulation. We also demonstrated that altering the intermediate amino acid did not alter the specificity of the regulatory proteins for their protein substrates.
Cite this paper
Tolentino, R. , Chastain, C. and Burnell, J. (2013) Identification of the amino acid involved in the regulation of bacterial pyruvate, orthophosphate dikinase and phosphoenolpyruvate synthetase. Advances in Biological Chemistry, 3, 12-21. doi: 10.4236/abc.2013.33A003.
Tolentino, R. , Chastain, C. and Burnell, J. (2013) Identification of the amino acid involved in the regulation of bacterial pyruvate, orthophosphate dikinase and phosphoenolpyruvate synthetase. Advances in Biological Chemistry, 3, 12-21. doi: 10.4236/abc.2013.33A003.
References
[1] Edwards, G.E., Nakamoto, H., Burnell, J.N. and Hatch, M.D. (1985) Pyruvate, Pi dikinase and NADP-malate dehydrogenase in C4 photosynthesis: Properties and mechanism of light/dark regulation. Annual Reviews of Plant Physiology, 36, 255-286. doi:10.1146/annurev.pp.36.060185.001351 [2] Hudspeth, R.L., Glackin C.A., Bonner J. and Grula J.W. (1986) Genomic and cDNA clones for maize phosphoenolpyruvate carboxylase and pyruvate, orthophosphate dikinase: Expression of different gene-family members in leaves and roots. Proceedings of the National Academy of Science USA, 83, 2884-2888. doi:10.1073/pnas.83.9.2884 [3] Sheen, J.-Y. and Bogorad, L. (1987) Regulation of levels of nuclear transcripts for C4 photo-synthesis in bundle sheath and mesophyll cells of maize leaves. Plant Molecular Biology, 8, 227-238. doi:10.1007/BF00015031 [4] Andrews T.J. and Hatch M.D. (1969) Properties and mechanism of action of pyruvate, phos-phate dikinase from leaves. Biochemical Journal, 114, 117-125. [5] Burnell, J.N. and Hatch, M.D. (1985) Light-dark modulation of leaf pyruvate, Pi dikinase. Trends in Biochemical Sciences, 111, 288-291. doi:10.1016/0968-0004(85)90090-8 [6] Burnell, J.N. and Hatch, M.D. (1984) Regulation of C4 photosynthesis: Catalytic phosphorylation as a prerequisite for ADP-mediated inactivation of pyruvate, Pi dikinase. Biochemical and Biophysical Research Communications, 118, 65-72. doi:10.1016/0006-291X(84)91068-4 [7] Ashton, A.R., Bur-nell, J.N. and Hatch, M.D. (1984) Regulation of C4 photosyn-thesis: Inactivation of pyruvate, Pidikinase by ADP-dependent phosphorylation and activation by phosphorolysis. Archives of Biochemistry and Biophysics, 230, 492-503. doi:10.1016/0003-9861(84)90429-6 [8] Burnell, J.N. and Hatch, M.D. (1983) Dark-light regulation of pyruvate, Pi diki-nase in C4 plants: Evidence that the same protein catalyses acti-vation and inactivation. Biochemical and Biophysical Research Communications, 111, 288-293. doi:10.1016/S0006-291X(83)80149-1 [9] Burnell, J.N. and Chastain, C.J. (2006) Cloning and expression of maize-leaf pyruvate, Pi dikinase regulatory protein gene. Biochemical and Biophysical Research Communications, 345, 675-680. doi:10.1016/j.bbrc.2006.04.150 [10] Tjaden, B., Plagens, A., Dorr, C., Siebers, B. and Hensel, R. (2006) Phosphoenolpyru-vate synthetase and pyruvate, phosphate dikinase of Thermo-proteus tenax: Key pieces in the puzzle of archaeal carbohydrate metabolism. Molecular Microbiology, 60, 287-298. doi:10.1111/j.1365-2958.2006.05098.x [11] Burnell, J.N. (2010) Cloning and characterization of Escherichia coli DUF299; A bifunctional ADP-dependent kinase-Pi-dependent pyrophosphorylase from bacteria. BMC Biochemistry, 11, 1. doi:10.1186/1471-2091-11-1 [12] Reiland, S., Messerli, G., Baerenfaller, K., Gerrits, B., Endler, A., Grossmann, J., Gruis-sem, W. and Baginsky, S. (2009) Large-scale Arabidopsis-phosphoproteome profiling reveals novel chloroplast kinase substrates and phosphorylation networks. Plant Physiology, 150, 889-903. doi:10.1104/pp.109.138677 [13] Bartlett, S., Seeliger, J. and Burnell, J.N. (2012) Identification of critical residues in the bifunctional phosphorenolpyruvate synthetase ki-nase/phosphotransferase of Escherichia coli. Current Topics in Biochemical Research, 14, 1. [14] Chastain, C.J., Botschner, M., Harrington, G.S., Thompson, B.J., Mills, S.E., Sarath, G. and Chollet, R. (2000) Further analysis of maize C4-pyruvate, orthophosphate dikinase phosphorylation by its bifunctional regulatory protein using selective substitutions of the regulatory Thr456 and catalytic His-458 residues. Archives of Biochemistry and Biophysics, 375, 165-170. doi:10.1006/abbi.1999.1651 [15] Chastain, C.J., Fries, J.P., Vogel, J.A., Randklev, C.L., Vossen, A.P., Dittmer, S.K., Watkins, E.E., Fiedler, L.J., Wacker, S.A., Meinhover, K.C., Sarath, G. and Chollet, R. (2002) Pyruvate, orthophosphate dikinase in leaves and chloroplasts of C3 plants undergoes light-/dark-induced reversible phosphorylation. Plant Physiology, 128, 13681378. doi:10.1104/pp.010806 [16] Burnell, J.N. (1984) Regulation of C4 photosynthesis: catalytic dephospho-rylation and Pi-mediated activation of pyruvate, Pi dikinase. Biochemical and Biophysical Research Communications, 120, 559-565. doi:10.1016/0006-291X(84)91291-9 [17] Chastain, C.J. and Chollet, R. (2003) Regulation of pyruvate, orthophosphate dikinase by ADP-/Pi-dependent reversible phosphorylation in C3 and C4 plants. Plant Physiology and Biochemistry, 41, 523-532. doi:10.1016/S0981-9428(03)00065-2 [18] Burnell, J.N. (1984) Regulation of C4 photosynthesis: proximity of the catalytically important histidine and the regulatory important threonine re-sidue of pyruvate, Pi dikinase. Biochemistry International, 9, 683-689. [19] Burnell, J.N. and Hatch, M.D. (1985) Regulation of C4 photosynthesis: Purification and properties of the protein catalyzing ADP-mediated inactivation and Pi-mediated activation of pyruvate, Pi dikinase. Archives of Biochemistry Biophysics, 237, 490-503. doi:10.1016/0003-9861(85)90302-9 [20] Astley, H., Parsley, K., Aubry, S.,Chastain, C.J., Burnell, J.N., Webb, M.E. and Hibberd, J.M. (2011) The pyruvate, orthophosphate dikinase regulatory proteins of Arabidopsis are both functional and in-teract with the catalytic and nucleotide binding domains of pyruvate, orthophosphate dikinase. The Plant Journal, 68, 1070-1081. doi:10.1111/j.1365-313X.2011.04759.x [21] Chulavatnatol, M. and Atkinson, D.E. (1973) Kinetic competition in vitro between phosphoenolpyruvate synthetase and the pyruvate dehydroge-nase complex from Escherichia coli. Journal of Biological Chemistry, 248, 27162721. [22] Varela-Gómez, M., More-no-Sánchez, R., Pardo, J.P. and Perez-Montfort, R. (2004) Ki-netic mechanism and metabolic role of pyruvate phosphate dikinase from Entamoebahistolytica. Journal of Biological Che-mistry, 279, 5412454130. doi:10.1074/jbc.M401697200 [23] Feng, X.M., Cao, L.J., Adam, R.D., Zhang, X.C. and Lu, S.Q. (2008) The catalyzing role of PPDK in Giardia lamblia. Biochemical and Biophysical Research Communications, 367, 394-398. doi:10.1016/j.bbrc.2007.12.139
[1] Edwards, G.E., Nakamoto, H., Burnell, J.N. and Hatch, M.D. (1985) Pyruvate, Pi dikinase and NADP-malate dehydrogenase in C4 photosynthesis: Properties and mechanism of light/dark regulation. Annual Reviews of Plant Physiology, 36, 255-286. doi:10.1146/annurev.pp.36.060185.001351 [2] Hudspeth, R.L., Glackin C.A., Bonner J. and Grula J.W. (1986) Genomic and cDNA clones for maize phosphoenolpyruvate carboxylase and pyruvate, orthophosphate dikinase: Expression of different gene-family members in leaves and roots. Proceedings of the National Academy of Science USA, 83, 2884-2888. doi:10.1073/pnas.83.9.2884 [3] Sheen, J.-Y. and Bogorad, L. (1987) Regulation of levels of nuclear transcripts for C4 photo-synthesis in bundle sheath and mesophyll cells of maize leaves. Plant Molecular Biology, 8, 227-238. doi:10.1007/BF00015031 [4] Andrews T.J. and Hatch M.D. (1969) Properties and mechanism of action of pyruvate, phos-phate dikinase from leaves. Biochemical Journal, 114, 117-125. [5] Burnell, J.N. and Hatch, M.D. (1985) Light-dark modulation of leaf pyruvate, Pi dikinase. Trends in Biochemical Sciences, 111, 288-291. doi:10.1016/0968-0004(85)90090-8 [6] Burnell, J.N. and Hatch, M.D. (1984) Regulation of C4 photosynthesis: Catalytic phosphorylation as a prerequisite for ADP-mediated inactivation of pyruvate, Pi dikinase. Biochemical and Biophysical Research Communications, 118, 65-72. doi:10.1016/0006-291X(84)91068-4 [7] Ashton, A.R., Bur-nell, J.N. and Hatch, M.D. (1984) Regulation of C4 photosyn-thesis: Inactivation of pyruvate, Pidikinase by ADP-dependent phosphorylation and activation by phosphorolysis. Archives of Biochemistry and Biophysics, 230, 492-503. doi:10.1016/0003-9861(84)90429-6 [8] Burnell, J.N. and Hatch, M.D. (1983) Dark-light regulation of pyruvate, Pi diki-nase in C4 plants: Evidence that the same protein catalyses acti-vation and inactivation. Biochemical and Biophysical Research Communications, 111, 288-293. doi:10.1016/S0006-291X(83)80149-1 [9] Burnell, J.N. and Chastain, C.J. (2006) Cloning and expression of maize-leaf pyruvate, Pi dikinase regulatory protein gene. Biochemical and Biophysical Research Communications, 345, 675-680. doi:10.1016/j.bbrc.2006.04.150 [10] Tjaden, B., Plagens, A., Dorr, C., Siebers, B. and Hensel, R. (2006) Phosphoenolpyru-vate synthetase and pyruvate, phosphate dikinase of Thermo-proteus tenax: Key pieces in the puzzle of archaeal carbohydrate metabolism. Molecular Microbiology, 60, 287-298. doi:10.1111/j.1365-2958.2006.05098.x [11] Burnell, J.N. (2010) Cloning and characterization of Escherichia coli DUF299; A bifunctional ADP-dependent kinase-Pi-dependent pyrophosphorylase from bacteria. BMC Biochemistry, 11, 1. doi:10.1186/1471-2091-11-1 [12] Reiland, S., Messerli, G., Baerenfaller, K., Gerrits, B., Endler, A., Grossmann, J., Gruis-sem, W. and Baginsky, S. (2009) Large-scale Arabidopsis-phosphoproteome profiling reveals novel chloroplast kinase substrates and phosphorylation networks. Plant Physiology, 150, 889-903. doi:10.1104/pp.109.138677 [13] Bartlett, S., Seeliger, J. and Burnell, J.N. (2012) Identification of critical residues in the bifunctional phosphorenolpyruvate synthetase ki-nase/phosphotransferase of Escherichia coli. Current Topics in Biochemical Research, 14, 1. [14] Chastain, C.J., Botschner, M., Harrington, G.S., Thompson, B.J., Mills, S.E., Sarath, G. and Chollet, R. (2000) Further analysis of maize C4-pyruvate, orthophosphate dikinase phosphorylation by its bifunctional regulatory protein using selective substitutions of the regulatory Thr456 and catalytic His-458 residues. Archives of Biochemistry and Biophysics, 375, 165-170. doi:10.1006/abbi.1999.1651 [15] Chastain, C.J., Fries, J.P., Vogel, J.A., Randklev, C.L., Vossen, A.P., Dittmer, S.K., Watkins, E.E., Fiedler, L.J., Wacker, S.A., Meinhover, K.C., Sarath, G. and Chollet, R. (2002) Pyruvate, orthophosphate dikinase in leaves and chloroplasts of C3 plants undergoes light-/dark-induced reversible phosphorylation. Plant Physiology, 128, 13681378. doi:10.1104/pp.010806 [16] Burnell, J.N. (1984) Regulation of C4 photosynthesis: catalytic dephospho-rylation and Pi-mediated activation of pyruvate, Pi dikinase. Biochemical and Biophysical Research Communications, 120, 559-565. doi:10.1016/0006-291X(84)91291-9 [17] Chastain, C.J. and Chollet, R. (2003) Regulation of pyruvate, orthophosphate dikinase by ADP-/Pi-dependent reversible phosphorylation in C3 and C4 plants. Plant Physiology and Biochemistry, 41, 523-532. doi:10.1016/S0981-9428(03)00065-2 [18] Burnell, J.N. (1984) Regulation of C4 photosynthesis: proximity of the catalytically important histidine and the regulatory important threonine re-sidue of pyruvate, Pi dikinase. Biochemistry International, 9, 683-689. [19] Burnell, J.N. and Hatch, M.D. (1985) Regulation of C4 photosynthesis: Purification and properties of the protein catalyzing ADP-mediated inactivation and Pi-mediated activation of pyruvate, Pi dikinase. Archives of Biochemistry Biophysics, 237, 490-503. doi:10.1016/0003-9861(85)90302-9 [20] Astley, H., Parsley, K., Aubry, S.,Chastain, C.J., Burnell, J.N., Webb, M.E. and Hibberd, J.M. (2011) The pyruvate, orthophosphate dikinase regulatory proteins of Arabidopsis are both functional and in-teract with the catalytic and nucleotide binding domains of pyruvate, orthophosphate dikinase. The Plant Journal, 68, 1070-1081. doi:10.1111/j.1365-313X.2011.04759.x [21] Chulavatnatol, M. and Atkinson, D.E. (1973) Kinetic competition in vitro between phosphoenolpyruvate synthetase and the pyruvate dehydroge-nase complex from Escherichia coli. Journal of Biological Chemistry, 248, 27162721. [22] Varela-Gómez, M., More-no-Sánchez, R., Pardo, J.P. and Perez-Montfort, R. (2004) Ki-netic mechanism and metabolic role of pyruvate phosphate dikinase from Entamoebahistolytica. Journal of Biological Che-mistry, 279, 5412454130. doi:10.1074/jbc.M401697200 [23] Feng, X.M., Cao, L.J., Adam, R.D., Zhang, X.C. and Lu, S.Q. (2008) The catalyzing role of PPDK in Giardia lamblia. Biochemical and Biophysical Research Communications, 367, 394-398. doi:10.1016/j.bbrc.2007.12.139