Abstract
Key message
NADP-ME2 from Arabidopsis thaliana exhibits a distinctive and complex regulation by fumarate, acting as an activator or an inhibitor according to substrate and effector concentrations. In this work, we used molecular modeling approach and site-directed mutagenesis to characterized the NADP-ME2 structural determinants necessary for allosteric regulation providing new insights for enzyme optimization.
Abstract
Structure–function studies contribute to deciphering how small modifications in the primary structure could introduce desirable characteristics into enzymes without affecting its overall functioning. Malic enzymes (ME) are ubiquitous and responsible for a wide variety of functions. The availability of a high number of ME crystal structures from different species facilitates comparisons between sequence and structure. Specifically, the structural determinants necessary for fumarate allosteric regulation of ME has been of particular interest. NADP-ME2 from Arabidopsis thaliana exhibits a distinctive and complex regulation by fumarate, acting as an activator or an inhibitor according to substrate and effector concentrations. However, the 3D structure for this enzyme is not yet reported. In this work, we characterized the NADP-ME2 allosteric site by structural modeling, molecular docking, normal mode analysis and mutagenesis. The regulatory site model and its docking analysis suggested that other C4 acids including malate, NADP-ME2 substrate, could also fit into fumarate’s pocket. Besides, a non-conserved cluster of hydrophobic residues in the second sphere of the allosteric site was identified. The substitution of one of those residues, L62, by a less flexible residue as tryptophan, resulted in a complete loss of fumarate activation and a reduction of substrate affinities for the active site. In addition, normal mode analysis indicated that conformational changes leading to the activation could originate in the region surrounding L62, extending through the allosteric site till the active site. Finally, the results in this work contribute to the understanding of structural determinants necessary for allosteric regulation providing new insights for enzyme optimization.
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Acknowledgements
We thank Dra. Ana Bortolotti for her help with the use of the fluorometer and the IBR Institute for lending us this equipment. A MCGW, MFD and CEA belong to the Researcher Career of National Council of Scientific and Technical Research (CONICET); CLA participated as a fellow of the same institution. AMTS and CRR thanks Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).
Funding
This work was supported by CONICET and National Agency for Promotion of Science and Technology. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001 (JFRM). This work was supported in part by a grant from Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Number E-26/203.179/2016.
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CEA conceived and led the project and together with MCGW and MFD designed and analyzed the experimental assays. CEA, CLA and MCGW designed the mutants and performed the kinetic and structural experiments. NCD performed and analyzed the NMA. JFRM and AMTS performed and analyzed the model structures and docking studies. CRR and AMTS supported all the computational work. All authors contributed to the writing of the manuscript and approved it.
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Supplementary file1 (PNG 3015 KB)
Supplementary Figure S1. Sequence alignment of A. thaliana NADP-ME2 and HsmNAD(P)-ME. In light blue is shown the N-terminal residues modeled with I-TASSER; in red is highlighted the residues analyzed in this work and in pink are non-conservative substitution.
Supplementary file2 (PNG 1016 KB)
Supplementary Figure S2. ME2-L62W purification and initial characterization protocol. (a) SDS-PAGE of aliquots from His-trap purification stages: Escherichia coli protein extract (1), pellet (2) and soluble fraction (3) after centrifugation, binding (4), flow through (5), wash (6) and elute after enterokinase digestion (7). (b) Native PAGE comparing to NADP-ME2 (3-5 mU of each protein were loaded). Molecular weight markers were run in parallel in both gels (MW). (c) Elution profile by gel filtration chromatography. The elution volume corresponds to a native molecular mass of 277 kDa according to the calibration curve shown. (d) Circular dichroism spectra of mutant and parental proteins.
Supplementary file3 (AVI 7827 KB)
Video 1. 3D overview of NADP-ME2 twisting motion. In the video is shown the twisting motion and the region (11´´-residues 60-80, highlighted in red) proposed to be responsible of allosteric signal transmission.
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Gerrard Wheeler, M.C., Arias, C.L., e Mello, J.F.R. et al. Structural insights into the allosteric site of Arabidopsis NADP-malic enzyme 2: role of the second sphere residues in the regulatory signal transmission. Plant Mol Biol 107, 37–48 (2021). https://doi.org/10.1007/s11103-021-01176-2
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DOI: https://doi.org/10.1007/s11103-021-01176-2