Phosphoenolpyruvate carboxylase

From Proteopedia

Function

Phosphoenolpyruvate carboxylase (PEPC) catalyzes the irreversible β-carboxylation of PEP in the presence of HCO3- to yield OAA (oxaloacetate) and Pi ( inorganic phosphate), using Mg2 + as a cofactor ^[1]. It is a key element in C4 carbon fixation ^[2].

Figure 1 Reaction catalyzed by PEPC. In the reaction, a carbon-carbon bond is formed, resulting in a product with four carbon atoms ("C4").

Overview

Figure 2 Diagrammatic representation of the C4 photosynthetic pathway (source)

The enzyme phosphoenolpyruvate carboxylase (PEPC) catalyzes the carboxylation of phosphoenolpyruvate to form oxaloacetate, with Mg2+ or Mn2+ as essential cofactors ^[3][4]. It can be considered the key enzyme in the C4 photosynthesis process, once it’s a central part of the mechanism that makes C4 plants more efficient in carbon fixation compared to classical C3-photosynthetic pathway plants, especially in abiotic stress environments ^[5]. PEPC is a ubiquitous enzyme, present in the genome of all plants. However, the isoforms found in C4 metabolism plants differ in their kinetic and regulatory characteristics, when compared to C3 orthologs ^[6]. Among the Flaverina genus of the Asteraceae family, closely related C3 and C4 species are found, providing a good model to study the differences between the two processes. The comparative analysis of Flaverina pringlei (C3) and Flaverina trinervia (C4) PEPC’s, has shown that the exchange of single amino acids can be responsible for the observed differences in saturation kinetics and inhibitor tolerance between PEPC’s of C3 and C4 species ^[7][6].

PEPC and C4 photosynthesis

In the classical C3 photosynthetic pathway, the RuBisCO enzyme, abundant in leaf mesophyll cells, is responsible for the carboxylation ribulose-1,5-bisphosphate (RuBP) and, therefore, for the assimilation of atmospheric CO2 into 3-phosphoglyceric acid (PGA). However, under high temperature or high concentrations of O2, RuBisCO’s oxygenating activity is favored leading to the oxygenation of RuBP, which produces phosphoglycolate (PG) as well as PGA. While PGA can readily be recycled back to RuBP via the Calvin cycle, PG has to be first metabolized into pyruvate and then to PGA. This process is commonly referred to as photorespiration and it leads to significant losses in freshly assimilated carbon to the atmosphere, essentially diminishing photosynthetic efficiency ^[8][5].

Plants with C4 photosynthetic metabolism, on the other hand, show very low levels of photorespiration, and consequently are more efficient in terms of carbon fixation ^[8]. That is made possible by a complex reorganization of leaf anatomy and metabolism in a CO2-concentrating mechanism that inhibits the photorespiratory pathway ^[5][6]. Without this characteristics, efficient carbon fixation in the mesophyll and subsequent carbon concentration in the BS wouldn’t be possible. Therefore, the evolution of PEPC’s C4 isoforms is one of the most important steps in the establishment of the C4 pathway ^[2].

spinqualitylabelsall models Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image PEPC enzyme structure Overall, plant PEPC’s present a similar structure characterized by four 105-110kDa identical subunits with a conserved N-terminal serine-phosphorylation domain, forming homotetramers [3][1]. X-ray crystallography studies on Escherichia coli and maize (Zea mays) PEPC’s show that the tetramer is comprised of two pairs of monomers with a greater amount of intersubunit contacts, suggesting a homotetrameric structure of a ‘‘dimer-of-dimers’’. The dimers are held together through an interaction between Arg-438 of one subunit and Glu-433 of the neighboring subunit, forming a salt bridge [9]. The monomeric structure maize’s C4-PEPC monomer consists of an eight-stranded β barrel and 42 α helices [3][10]. The of each monomer, bound to PEP and the cofactor, is located in the C-terminus region of the β barrel [11]. Figure 3 X-ray crystal structure of Flaverina trinervia’s C4 PEPC bound to glucose 6-phosphate (magenta). Two strongly bound dimers (left and right sides of the structure) form the tetrameric quaternary structure. Adapted from Schlieper et al. 2014. [12] The comparison between closely related C3 and C4 PEPC’s from F. pringlei (C3) and F. trinervia (C4) was very important in determining the changes responsible for the differences in the enzymes activity in C3 and C4 plants. Structural superposition of these two isoforms shows high levels of structural similarity, supported by the low backbone root-mean square deviation of 0.4 Å [6]. Two important site-specific differences between the two structures are a substitution of the 884 residue located close to the feedback inhibitor-binding site, and another residue substitution at the 774 position. The first is largely responsible for the drastic differences observed between the inhibitor tolerances of the C3 and C4 PEPC’s, while the second is a key determinant for the different kinetic properties of F. pringlei and F. trinervia PEPC’s [7][6]. Allosteric regulation and reaction mechanism PEPC’s carboxylase activity is regulated by different post-translational mechanisms. In C4 and CAM plants, the phosphorylation of a serine residue near the N terminus (S15 in maize C4-PEPC) activates the enzyme by decreasing its sensitivity to allosteric inhibitors such as aspartate and malate and increasing activation by the positive allosteric regulator glucose 6-phosphate [1]. Studies on F. pringlei and F. trinervia, have positively identified the residues Arg641, Lys829, Arg888 and Asn964 as binding motif of the negative allosteric inhibitors aspartate and malate [6]. Similar studies have also identified the site in maize [11]. In C3 PEPC, Arg884 provides an additional hydrogen bond for inhibitor binding, whereas in C4 PEPC isoforms the substitution of this residue by a glycine, reducing the enzymes sensitivity towards both feedback inhibitors [13][6]. The positive allosteric effector glucose 6-phosphate’s binding site has also been identified in the C4-PEPC of maize. X-ray crystallography of maize’s C4-PEPC in complex with sulfate ion (a positive effector analog of glucose 6-phosphate) revealed that the positive effector was bound to the enzyme at the dimer interface and was surrounded by four positively charged residues (R183, R184, R231, and R372 in the adjacent subunit [3]. Figure 4 Inhibitor-binding site of Flaverina trinervia’s C4 PEPC. Adapted from Paulus et al. 2013. [6] Most proposed catalytic reaction mechanisms suggest that PEPC catalyzes an ordered multistep reaction in which the preferred order of reactant binding to the active site are: first the bivalent cation ( Mg2+ or Mn2+), then PEP and lastly bicarbonate (HCO3- ) [3][10]. In a review article on the subject, Izui et al., 2004 [10], proposes a detailed reaction mechanism for the enzyme, based on maize and E.coli PEPC structures in which PEP is located in a hydrophobic pocket consisting of W248, L504, and M538 residues. In this model, H177 is a critically important catalytic base as it is supposed to play a role in stabilizing the carboxyphosphate intermediate and abstracting a proton from its carboxyl group. Figure 5 Catalytic mechanism of PEPC based on maize C4-PEPC and E. coli PEPC crystal structures and the three-step reaction model. The hydrophobic pocket is shown as yellow circles and the residues show maize numbering. Adapted from Izui et al. 2004. [10]

3D structures of phosphoenolpyruvate carboxylase

Escherichia coli
1jqn – EcPEPC + aspartate + PEP analog
1qb4 – EcPEPC + aspartate
1fiy – EcPEPC + aspartate

Zea mays
1jqo – ZmPEPC
6v3o – ZmPEPC + citrate
5vyj – ZmPEPC + glycine
6u2t – ZmPEPC + malate
6mgi – ZmPEPC + α-D-glucose-6-phosphate

Flaverina trinervia
4bxc – FtPEPC + α-D-glucose-6-phosphate
4bxh – FtPEPC

Arabidopsis thaliana
8oj9 – AtPEPC 1
8ojf – AtPEPC 1 + phosphate
8oje – AtPEPC 1 + malate
5fdn – AtPEPC 3 + aspartate + citrate

Flaveria pringlei
3zge – FpPEPC + aspartate
3zgb – FpPEPC + aspartate

Actinomyces israelii
6k31 – PEPC + CO

Clostridium perfringens
3odm – PEPC + malonate

Updated on 24-August-2023

References

1. ↑ ^{1.0 1.1 1.2} O'Leary B, Park J, Plaxton WC. The remarkable diversity of plant PEPC (phosphoenolpyruvate carboxylase): recent insights into the physiological functions and post-translational controls of non-photosynthetic PEPCs. Biochem J. 2011 May 15;436(1):15-34. PMID:21524275 doi:10.1042/BJ20110078
2. ↑ ^{2.0 2.1} Sage RF. The evolution of C(4) photosynthesis. New Phytol. 2004 Feb;161(2):341-370. PMID:33873498 doi:10.1111/j.1469-8137.2004.00974.x
3. ↑ ^{3.0 3.1 3.2 3.3 3.4} Kai Y, Matsumura H, Izui K. Phosphoenolpyruvate carboxylase: three-dimensional structure and molecular mechanisms. Arch Biochem Biophys. 2003 Jun 15;414(2):170-9. PMID:12781768 doi:10.1016/s0003-9861(03)00170-x
4. ↑ Svensson P, Bläsing OE, Westhoff P. Evolution of C4 phosphoenolpyruvate carboxylase. Arch Biochem Biophys. 2003 Jun 15;414(2):180-8. PMID:12781769 doi:10.1016/s0003-9861(03)00165-6
5. ↑ ^{5.0 5.1 5.2} Sage RF, Sage TL, Kocacinar F. Photorespiration and the evolution of C4 photosynthesis. Annu Rev Plant Biol. 2012;63:19-47. PMID:22404472 doi:10.1146/annurev-arplant-042811-105511
6. ↑ ^{6.0 6.1 6.2 6.3 6.4 6.5 6.6 6.7} Paulus JK, Schlieper D, Groth G. Greater efficiency of photosynthetic carbon fixation due to single amino-acid substitution. Nat Commun. 2013 Feb 26;4:1518. doi: 10.1038/ncomms2504. PMID:23443546 doi:http://dx.doi.org/10.1038/ncomms2504
7. ↑ ^{7.0 7.1} Bläsing OE, Westhoff P, Svensson P. Evolution of C4 phosphoenolpyruvate carboxylase in Flaveria, a conserved serine residue in the carboxyl-terminal part of the enzyme is a major determinant for C4-specific characteristics. J Biol Chem. 2000 Sep 8;275(36):27917-23. PMID:10871630 doi:10.1074/jbc.M909832199
8. ↑ ^{8.0 8.1} Bauwe, H. Chapter 6 Photorespiration: The Bridge to C4 Photosynthesis. in C4 Photosynthesis and Related CO2 Concentrating Mechanisms (eds. Raghavendra, A. S. & Sage, R. F.) 81–108 (Springer Netherlands, 2011). doi:10.1007/978-90-481-9407-0_6.
9. ↑ Kai Y, Matsumura H, Inoue T, Terada K, Nagara Y, Yoshinaga T, Kihara A, Tsumura K, Izui K. Three-dimensional structure of phosphoenolpyruvate carboxylase: a proposed mechanism for allosteric inhibition. Proc Natl Acad Sci U S A. 1999 Feb 2;96(3):823-8. PMID:9927652
10. ↑ ^{10.0 10.1 10.2 10.3} Izui K, Matsumura H, Furumoto T, Kai Y. Phosphoenolpyruvate carboxylase: a new era of structural biology. Annu Rev Plant Biol. 2004;55:69-84. PMID:15725057 doi:10.1146/annurev.arplant.55.031903.141619
11. ↑ ^{11.0 11.1} Matsumura H, Xie Y, Shirakata S, Inoue T, Yoshinaga T, Ueno Y, Izui K, Kai Y. Crystal structures of C4 form maize and quaternary complex of E. coli phosphoenolpyruvate carboxylases. Structure. 2002 Dec;10(12):1721-30. PMID:12467579
12. ↑ Schlieper D, Foerster K, Paulus JK, Groth G. Resolving the activation site of positive regulators in plant phosphoenolpyruvate carboxylase. Mol Plant. 2013 Sep 16. PMID:24043710 doi:10.1093/mp/sst130
13. ↑ Dharmarajan L, Kraszewski JL, Mukhopadhyay B, Dunten PW. Structure of an archaeal-type phosphoenolpyruvate carboxylase sensitive to inhibition by aspartate. Proteins. 2011 Feb 3. doi: 10.1002/prot.23006. PMID:21491491 doi:10.1002/prot.23006