ATP-dependent phosphoenolpyruvate carboxykinase (PEPCK) is a key catabolic enzyme found in various species of bacteria, plants, and yeast. PEPCK may play a role in carbon fixation in aquatic ecosystems consisting of photosynthetic cyanobacteria. RuBisCO-based CO2 fixation is prevalent in cyanobacteria through C3 intermediates; however, a significant amount of carbon flows into C4 acids during cyanobacterial photosynthesis. This indicates that a C4 mechanism for inorganic carbon fixation is prevalent in cyanobacteria with PEPCK as an important β-carboxylation enzyme. Newly available genomic information has confirmed the existence of putative PEPCK genes in a number of cyanobacterial species. This project represents the first structural and physicochemical study of cyanobacterial PEPCKs. Biocomputational analyses of cyanobacterial PEPCKs were performed and a homology model of Cyanothece sp. PCC 7424 PEPCK was generated. The modeled enzyme consists of an N-terminal and C-terminal domains with a mixed α/β topology with the active site located in a deep cleft between the two domains. Active site residues and those involved in metal ion coordination were found to be conserved in the cyanobacterial enzymes. An active site lid which is known to close upon substrate binding was also predicted. Amino acid stretches that are unique to cyanobacterial PEPCKs were also identified. 1. Introduction Phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.32) catalyzes the reversible ATP- or GTP-dependent decarboxylation of oxaloacetate (OAA) to yield phosphoenolpyruvate (PEP). This reaction uses the phosphate group from the nucleotide triphosphate and, as a result, produces CO2 and the corresponding nucleoside diphosphate. PEPCK has a strict requirement for divalent cations with Mn2+ as its best activator [1]. Two classes of PEPCKs exist in nature, and they are classified in the basis of the nucleotide substrate: ATP-utilizing enzymes are found in bacteria, plants, and yeast, while GTP-dependent PEPCKs are found mostly in higher eukaryotes [2]. GTP-dependent PEPCKs also occur in some bacteria such as Corynebacterium glutamicum [3]. While there is no significant sequence identity between the two classes, a number of residues are completely conserved across all PEPCKs in the regions of the enzyme that are necessary for nucleotide binding and metal ion coordination [1]. The crystal structures of PEPCKs from representative species of plants, bacteria, and mammals have been published, and conservation in metal and substrate binding were confirmed [4]. In mammals, two forms of
References
[1]
G. M. Carlson and T. Holyoak, “Structural insights into the mechanism of phosphoenolpyruvate carboxykinase catalysis,” Journal of Biological Chemistry, vol. 284, no. 40, pp. 27037–27041, 2009.
[2]
P. Dunten, C. Belunis, R. Crowther et al., “Crystal structure of human cytosolic phosphoenolpyruvate carboxykinase reveals a new GTP-binding site,” Journal of Molecular Biology, vol. 316, no. 2, pp. 257–264, 2002.
[3]
S. Aich, L. Prasad, and L. T. J. Delbaere, “Structure of a GTP-dependent Bacterial PEP-carboxykinase from Corynebacterium glutamicum,” International Journal of Biochemistry and Cell Biology, vol. 40, no. 8, pp. 1597–1603, 2008.
[4]
Y. A. Leduc, L. Prasad, M. Laivenieks, J. G. Zeikus, and L. T. J. Delbaere, “Structure of PEP carboxykinase from the succinate-producing Actinobacillus succinogenes: a new conserved active-site motif,” Acta Crystallographica Section D, vol. 61, no. 7, pp. 903–912, 2005.
[5]
M. A. Larkin, G. Blackshields, N. P. Brown et al., “Clustal W and Clustal X version 2.0,” Bioinformatics, vol. 23, no. 21, pp. 2947–2948, 2007.
[6]
E. Gasteiger, C. Hoogland, A. Gattiker et al., “Protein identification and analysis tools on the ExPASy server,” in The Proteomics Protocols Handbook, J. M. Walker, Ed., pp. 571–607, Humana Press, 2005.
[7]
J. Yang, S. C. Kalhan, and R. W. Hanson, “What is the metabolic role of phosphoenolpyruvate carboxykinase?” Journal of Biological Chemistry, vol. 284, no. 40, pp. 27025–27029, 2009.
[8]
N. Asanuma, K. Kanada, Y. Arai, K. Yoshizawa, T. Ichikawa, and T. Hino, “Molecular characterization and significance of phosphoenolpyruvate carboxykinase in a ruminal bacterium, streptococcus bovis,” Journal of General and Applied Microbiology, vol. 56, no. 2, pp. 121–127, 2010.
[9]
Y. D. Kwon, S. Y. Lee, and P. Kim, “A physiology study of Escherichia coli overexpressing phosphoenolpyruvate carboxykinase,” Bioscience, Biotechnology and Biochemistry, vol. 72, no. 4, pp. 1138–1141, 2008.
[10]
R. M. Zelle, J. Trueheart, J. C. Harrison, J. T. Pronk, and A. J. A. Van Maris, “Phosphoenolpyruvate carboxykinase as the sole anaplerotic enzyme in saccharomyces cerevisiae,” Applied and Environmental Microbiology, vol. 76, no. 16, pp. 5383–5389, 2010.
[11]
R. P. Walker, Z. H. Chen, R. M. Acheson, and R. C. Leegood, “Effects of phosphorylation on phosphoenolpyruvate carboxykinase from the C4 plant Guinea grass,” Plant Physiology, vol. 128, no. 1, pp. 165–172, 2002.
[12]
M. Martín, S. P. Rius, and F. E. Podestá, “Two phosphoenolpyruvate carboxykinases coexist in the Crassulacean Acid Metabolism plant Ananas comosus. Isolation and characterization of the smaller 65kDa form,” Plant Physiology and Biochemistry, vol. 49, no. 6, pp. 646–653, 2011.
[13]
J. R. Reinfelder, A. J. Milligan, and F. M. M. Morel, “The role of the C4 pathway in carbon accumulation and fixation in a marine diatom,” Plant Physiology, vol. 135, no. 4, pp. 2106–2111, 2004.
[14]
J. Xu, X. Fan, X. Zhang et al., “Evidence of coexistence of C3 and C4 photosynthetic pathways in a green-tide-forming alga, Ulva prolifera,” PLoS ONE, vol. 7, no. 5, Article ID e37438, 2012.
[15]
A. A. Smith, M. W. Coomes, and T. E. Smith, “Isolation and sequence of the phosphoenolpyruvate carboxylase gene of the marine cyanobacterium Synechococcus PCC 7002,” Journal of Biological Sciences, vol. 8, no. 8, pp. 1261–1270, 2008.
[16]
B. O'Leary, S. K. Rao, J. Kim, and W. C. Plaxton, “Bacterial-type phosphoenolpyruvate carboxylase (PEPC) functions as a catalytic and regulatory subunit of the novel class-2 PEPC complex of vascular plants,” Journal of Biological Chemistry, vol. 284, no. 37, pp. 24797–24805, 2009.
[17]
S. R. Smith, R. M. Abbriano, and M. Hildebrand, “Comparative analysis of diatom genomes reveals substantial differences in the organization of carbon partitioning pathways,” Algal Research, vol. 1, no. 1, pp. 2–16, 2012.
[18]
A. A. Smith and M. C. Plazas, “In silico characterization and homology modeling of cyanobacterial Phosphoenolpyruvate carboxylase enzymes with computational tools and bioinformatics servers,” American Journal of Biochemistry and Molecular Biology, vol. 1, no. 4, pp. 319–336, 2011.
[19]
D. Gilbert, “Sequence file format conversion with command-line readseq,” in Current Protocols in Bioinformatics, A. D. Baxevanis, G. A. Petsko, L. D. Stein, and G. D. Stormo, Eds., John Wiley & Sons, Hoboken, NJ, USA, 2003.
[20]
R. A. Laskowski, V. V. Chistyakov, and J. M. Thornton, “PDBsum more: new summaries and analyses of the known 3D structures of proteins and nucleic acids,” Nucleic Acids Research, vol. 33, pp. D266–D268, 2005.
[21]
K. Arnold, L. Bordoli, J. Kopp, and T. Schwede, “The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling,” Bioinformatics, vol. 22, no. 2, pp. 195–201, 2006.
[22]
S. C. Lovell, I. W. Davis, W. B. Arendall et al., “Structure validation by Cα geometry: φ,ψ and Cβ deviation,” Proteins: Structure, Function and Genetics, vol. 50, no. 3, pp. 437–450, 2003.
P. Rice, L. Longden, and A. Bleasby, “EMBOSS: the European molecular biology open software suite,” Trends in Genetics, vol. 16, no. 6, pp. 276–277, 2000.
[25]
F. Eisenhaber, F. Imperiale, P. Argos, and C. Frommel, “Prediction of secondary structural content of proteins from their amino acid composition alone. I. New analytic vector decomposition methods,” Proteins, vol. 25, no. 2, pp. 157–168, 1996.
[26]
N. Guex and M. C. Peitsch, “SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling,” Electrophoresis, vol. 18, no. 15, pp. 2714–2723, 1997.
[27]
G. Vriend, “WHAT IF: a molecular modeling and drug design program,” Journal of Molecular Graphics, vol. 8, no. 1, pp. 52–56, 1990.
[28]
P. Benkert, M. Biasini, and T. Schwede, “Toward the estimation of the absolute quality of individual protein structure models,” Bioinformatics, vol. 27, no. 3, pp. 343–350, 2011.
[29]
C. Andrade, C. Sepulveda, E. Cardemil, and A. M. Jabalquinto, “The role of tyrosine 207 in the reaction catalyzed by Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase,” Biological Research, vol. 43, no. 2, pp. 191–195, 2010.
[30]
M. Sugahara, N. Ohshima, Y. Ukita, M. Sugahara, and N. Kunishima, “Structure of ATP-dependent phosphoenolpyruvate carboxykinase from Thermus thermophilus HB8 showing the structural basis of induced fit and thermostability,” Acta Crystallographica Section D, vol. 61, no. 11, pp. 1500–1507, 2005.
[31]
S. Trapani, J. Linss, S. Goldenberg, H. Fischer, A. F. Craievich, and G. Oliva, “Crystal structure of the dimeric phosphoenolpyruvate carboxykinase (PEPCK) from Trypanosoma cruzi at 2 ？ resolution,” Journal of Molecular Biology, vol. 313, no. 5, pp. 1059–1072, 2001.
[32]
J. J. H. Cotelesage, L. Prasad, J. G. Zeikus, M. Laivenieks, and L. T. J. Delbaere, “Crystal structure of Anaerobiospirillum succiniciproducens PEP carboxykinase reveals an important active site loop,” International Journal of Biochemistry and Cell Biology, vol. 37, no. 9, pp. 1829–1837, 2005.
[33]
L. A. Kelley and M. J. Sternberg, “Protein structure prediction on the web: a case study using the Phyre server,” Nature Protocols, vol. 4, no. 3, pp. 363–371, 2009.
[34]
C. Lambert, N. Léonard, X. De Bolle, and E. Depiereux, “ESyPred3D: prediction of proteins 3D structures,” Bioinformatics, vol. 18, no. 9, pp. 1250–1256, 2002.
[35]
L. W. Tari, A. Matte, U. Pugazhenthi, H. Goldie, and L. T. J. Delbaere, “Snapshot of an enzyme reaction intermediate in the structure of the ATP-Mg2+-oxalate ternary complex of Escherichia coli PEP carboxykinase,” Nature Structural Biology, vol. 3, no. 4, pp. 355–363, 1996.
[36]
S. M. Sullivan and T. Holyoak, “Enzymes with lid-gated active sites must operate by an induced fit mechanism instead of conformational selection,” Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 37, pp. 13829–13834, 2008.
[37]
T. A. Johnson and T. Holyoak, “Increasing the conformational entropy of the Ω-loop lid domain in phosphoenolpyruvate carboxykinase impairs catalysis and decreases catalytic fidelity,” Biochemistry, vol. 49, no. 25, pp. 5176–5187, 2010.
