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Molecular adaptations of NADP-malic enzyme for its function in C4 photosynthesis in grasses


In C4 grasses of agronomical interest, malate shuttled into the bundle sheath cells is decarboxylated mainly by nicotinamide adenine dinucleotide phosphate (NADP)-malic enzyme (C4-NADP-ME). The activity of C4-NADP-ME was optimized by natural selection to efficiently deliver CO2 to Rubisco. During its evolution from a plastidic non-photosynthetic NADP-ME, C4-NADP-ME acquired increased catalytic efficiency, tetrameric structure and pH-dependent inhibition by its substrate malate. Here, we identified specific amino acids important for these C4 adaptions based on strict differential conservation of amino acids, combined with solving the crystal structures of maize and sorghum C4-NADP-ME. Site-directed mutagenesis and structural analyses show that Q503, L544 and E339 are involved in catalytic efficiency; E339 confers pH-dependent regulation by malate, F140 is critical for the stabilization of the oligomeric structure and the N-terminal region is involved in tetramerization. Together, the identified molecular adaptations form the basis for the efficient catalysis and regulation of one of the central biochemical steps in C4 metabolism.

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Fig. 1: Quaternary organization of C4-NADP-ME.
Fig. 2: Candidate amino acids for functional analysis identified using Zm- and SbC4-NADP-ME crystal structures.
Fig. 3: Comparison of ZmC4-NADP-ME variants shows that the identified amino acids contribute to the kinetic differences between C4- and nonC4 isoforms.
Fig. 4: Comparison of ZmnonC4-NADP-ME variants confirms pivotal roles for substitutions I140F and A339E in combination with an N-terminal deletion.
Fig. 5: F140, but none of the other strictly differentially conserved residues, contributes to stabilization of the tetramer.
Fig. 6: Positions and interactions of F140 and E339 in the C4-NADP-ME structure.

Data availability

The data supporting the findings of this manuscript are available from the corresponding author upon reasonable request. The protein crystallographic structures were deposited in the Protein Data Bank (wwpdb) under the accession codes 5OU5 (ZmC4-NADP-ME) and 6C7N (SbC4-NADP-ME).


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This work was funded by grants of the European Union (3to4) to V.G.M. and by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany´s Excellence Strategy (EXC 2048/1, Project ID: 390686111 and EXC 1028, to V.G.M. and M.J.L. We acknowledge the European Synchrotron Radiation Facility (ESRF) for provision of synchrotron radiation facilities and we wish to thank L. Gordon for assistance in using beamline ID23-1. The Center for Structural Biology of Mercosur granted funding to C.E.A. for data collection at Institut Pasteur de Montevideo (IPM). We are grateful to N. Larrieux at the Protein Crystallography Facility IPM for assistance with crystallization and data collection.

Author information




V.G.M. conceived and led the project and, together with M.F.D., designed and supervised the work and analysed data. C.-C.W., A.Bo. and M.S. produced the ZmC4- and ZmnonC4-NADP-ME recombinant proteins and obtained kinetic and structural data. A.H. planned crystallization studies of ZmC4-NADP-ME, collected X-ray diffraction data and solved, refined and analysed the structure. A.Bo. and A.H. produced ZmC4-NADP-ME crystals. C.E.A., F.T. and A.Bu. crystallized SbC4-NADP-ME, collected X-ray diffraction data and solved, refined and analysed the structure. C.-C.W. and M.J.L. designed the algorithm to identify strictly differentially conserved amino acid residues and performed the bioinformatic analyses. T.Z. and L.N.-S. performed and analysed circular dichroism and analytical ultracentrifugation analysis. All authors contributed equally to writing the manuscript and generation of the Figures.

Corresponding author

Correspondence to Veronica G. Maurino.

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The authors declare no competing interests.

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Peer review information: Nature Plants thanks Robert Furbank and Liang Tong and other, anonymous, reviewers for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Discussion, Supplementary Figs. 1–8, Supplementary Tables 1–4, Supplementary Video legends.

Reporting Summary

Supplementary Video 1

3D overview of the overall SbC4-NADP-ME structure and the position of the mutated amino acids. The first section (1´´–30´´) shows the overall structure with the active site in each monomer, and the tilted position of each dimer in the dimer–dimer quaternary structure. The second section (30´´–60´´) shows the 3D position of the four mutated amino acids (F140, E339, Q503 and L544).

Supplementary Video 2

3D view of the interface connection between monomers in SbC4-NADP-ME. The first section (1´´–30´´) shows the position of F140 in monomers A and B. The second section (30´´-60´´) shows the position of the N-terminal region in the contact interface between each monomer. The N termini were selected such that they range from amino acid 84, the initial residue obtained in the SbC4-NADP-ME crystal structure, to amino acid 102 (corresponding to the chimeric proteins produced in ref. 15). Only selected residues/moieties are shown for clarity.

Supplementary Video 3

3D view of putative malate allosteric binding site (0´´–40´´). Shown are different views of the region surrounding E339 with its possible points of connection with active sites in SbC4-NADP-ME and the open/close switch conformation that is essential for catalysis.

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Alvarez, C.E., Bovdilova, A., Höppner, A. et al. Molecular adaptations of NADP-malic enzyme for its function in C4 photosynthesis in grasses. Nat. Plants 5, 755–765 (2019).

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