Phosphoenolpyruvate carboxylase: Difference between revisions
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'''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 <ref name="O'Leary2011">PMID: 21524275</ref>. It is a key element in [https://en.wikipedia.org/wiki/C4_carbon_fixation C4 carbon fixation] <ref name="sage2004">PMID: 33873498</ref>. | '''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 <ref name="O'Leary2011">PMID: 21524275</ref>. It is a key element in [https://en.wikipedia.org/wiki/C4_carbon_fixation C4 carbon fixation] <ref name="sage2004">PMID: 33873498</ref>. | ||
[[Image:C4_PEPC_reaction_and_regulation.png|center|800px | [[Image:C4_PEPC_reaction_and_regulation.png|center|thumb|800px|caption position=bottom|'''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 == | == Overview == | ||
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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"/>. | 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"/>. | ||
<StructureSection load='' size='400' side='right' scene='57/573979/Tetramer/ | <StructureSection load='' size='400' side='right' scene='57/573979/Tetramer/2' pspeed='8'> | ||
== PEPC enzyme structure == | == 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 <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 dimers are held together through an interaction between Arg-438 of one subunit and Glu-433 of the neighboring subunit, forming a salt bridge <ref>PMID: 9927652</ref>. 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 <scene name='57/573979/Cv/ | 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 dimers are held together through an interaction between Arg-438 of one subunit and Glu-433 of the neighboring subunit, forming a salt bridge <ref>PMID: 9927652</ref>. 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 <scene name='57/573979/Cv/3'>active site</scene> of each monomer, bound to PEP and the <scene name='57/573979/Cv/3'>Mn+2 ion</scene> cofactor, is located in the C-terminus region of the β barrel <ref name="matsumura2002">PMID: 12467579</ref>. | ||
[[Image:4BXC_F.trinervia_PEPC+G6P_crystral_structure.png|center|frame|caption position=bottom|'''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. <ref name="schlieper2014">PMID: 24043710</ref>]] | [[Image:4BXC_F.trinervia_PEPC+G6P_crystral_structure.png|center|frame|caption position=bottom|'''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. <ref name="schlieper2014">PMID: 24043710</ref>]] | ||
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== Allosteric regulation and reaction mechanism == | == 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 <ref name="O'Leary2011"/>. 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 <ref name="Paulus2013"/>. Similar studies have also identified the <scene name='57/573979/Cv/ | 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 ''F. trinervia'', have positively identified the residues Arg641, Lys829, Arg888 and Asn964 as binding motif of the negative allosteric inhibitors aspartate and malate <ref name="Paulus2013"/>. Similar studies have also identified the <scene name='57/573979/Cv/8'>aspartate binding site</scene> site in maize <ref name="matsumura2002"/>. 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"/>. | ||
[[Image:Inhibitor-binding_site_of_Flaverina_trinervia_C4_PEPC.png|center|frame|caption position=bottom|'''Figure 4''' Inhibitor-binding site of ''Flaverina trinervia’s'' C4 PEPC. Adapted from Paulus et al. 2013. <ref name="Paulus2013"/>]] | [[Image:Inhibitor-binding_site_of_Flaverina_trinervia_C4_PEPC.png|center|frame|caption position=bottom|'''Figure 4''' Inhibitor-binding site of ''Flaverina trinervia’s'' C4 PEPC. Adapted from Paulus et al. 2013. <ref name="Paulus2013"/>]] | ||
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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. | 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. | ||
[[Image:PEPC reaction mechanism.png|center| | [[Image:PEPC reaction mechanism.png|center|thumb|600 px|caption position=bottom|'''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. <ref name="Izui2004"/>]] | ||
</StructureSection> | </StructureSection> | ||
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'''''Arabidopsis thaliana'''''<br> | '''''Arabidopsis thaliana'''''<br> | ||
[[5fdn]] – AtPEPC 3 + aspartate + citrate<br> | [[8oj9]] – AtPEPC 1 <br /> | ||
[[8ojf]] – AtPEPC 1 + phosphate <br /> | |||
[[8oje]] – AtPEPC 1 + malate <br /> | |||
[[5fdn]] – AtPEPC 3 + aspartate + citrate <br /> | |||
'''''Flaveria pringlei'''''<br> | '''''Flaveria pringlei'''''<br> | ||