Phosphoenolpyruvate carboxylase: Difference between revisions

The enzyme phosphoenolpyruvate carboxylase (PEPC) catalyzes the carboxylation of phosphoenolpyruvate to form oxaloacetate, with Mg2+ or Mn2+ as essential cofactors <ref name="kai2003">PMID: 12781768</ref><ref>PMID: 12781769</ref>. 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  <ref name="sage2012">PMID: 22404472</ref>. 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  <ref name="Paulus2013">PMID: 23443546</ref>. 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 F. pringlei (C3) and T. 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 <ref name="blasing2000">PMID: 10871630</ref><ref name="Paulus2013"/>.

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 <ref name="bauwe2011">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.</ref><ref name="sage2012"/>.

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 <ref name="bauwe2011"/>. That is made possible by a complex reorganization of leaf anatomy and metabolism in a CO2-concentrating mechanism that inhibits the photorespiratory pathway <ref name="sage2012">. This type of plants present a distinguished leaf anatomy called Kranz anatomy, where CO2 and RuBisCO are concentrated in a distinct cell layer between the mesophyll cells and the vascular bundles, called bundle sheath (BS) tissue <ref name="sage2012"/>. In all C4 plants, carbon, in the form of bicarbonate, is initially fixed to phosphoenolpyruvate (PEP) in mesophyll cells by the  cytosolic enzyme PEPC forming a four-carbon organic acid, oxaloacetic acid (OAA), hence the name C4 photosynthesis. Still in the outer compartment, OAA is converted to malate (MAL), that than diffuses through plasmodesmata into the BS compartment, where it is decarboxylated by NADP-ME, releasing CO2, NADPH, and pyruvate (PVA). Through this process, the CO2 level can be concentrated by up to 10 times in the intercellular spaces inside the BS, thereby suppressing Rubisco’s oxygenase activity and carbon loss due to photorespiration <ref name="sage2004"/><ref name="sage2012">

Although all enzymes of the C4 cycle are present in C3 plants, there are important changes found in C4 isoforms, specially in PEPC enzymes, that make C4 metabolism possible. PEPC’s have several non-photosynthetic related functions in different plant tissues. In C4 plants, however, changes in the promotor and enzyme structures allow for strong and specific mesophyll expression, reduced sensitivity to negative feedback inhibitors and higher kinetic efficiency <ref name="blasing2000"/><ref>PMID: 12781769</ref><ref name="Paulus2013"/>. 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 <ref name="sage2004"/>.

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 <ref name="kai2003"/><ref name="O'Leary2011"/>. 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 monomeric structure  maize’s C4-PEPC monomer consists of an eight-stranded β barrel and  42 α helices <ref name="kai2003"/><ref name="Izui2004">PMID: 15725057</ref>.

The comparison between closely related C3 and C4 PEPC’s from F. pringlei (C3) and T. 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 <ref name="Paulus2013"/>.  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 T. trinervia PEPC’s <ref name="blasing2000"/><ref name="Paulus2013"/>.

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 <ref name="O'Leary2011"/>. Studies on F. pringlei and T. trinervia, have positively identified the residues Arg641, Lys829, Arg888 and Asn964 as binding motif of the negative allosteric inhibitors aspartate and malate. 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 <ref>PMID: 21491491</ref><ref name="Paulus2013"/>. 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 <ref name="kai2003"/>.

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- ) <ref name="kai2003"/><ref name="Izui2004"/>. In a review article on the subject, Izui et al., 2004 <ref name="Izui2004"/>, 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.