Multiple conformations in solution of the maize C4-phosphoenolpyruvate carboxylase isozyme

The photosynthetic phosphoenolpyruvate carboxylase isozyme from C4 plants (PEPC-C4) has a complex allosteric regulation, involving positive cooperativity in binding the substrate phosphoenolpyruvate as well as positive and negative allosteric effectors. Besides the proposed R- and T-states, previous...

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Autores principales:	Fátima Barreda-Huerta, Ismael Bustos-Jaimes, Carlos Mújica-Jiménez, Rosario A. Muñoz-Clares
Formato:	article
Lenguaje:	EN
Publicado:	Elsevier 2021
Materias:	Zea mays L. C4 metabolism Allosteric regulation Ligand-induced conformational changes ANS fluorescence CD spectroscopy Science (General) Q1-390 Social sciences (General) H1-99
Acceso en línea:	https://doaj.org/article/d3ec3990da20405cbf99c3d25883b6bd
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spelling	oai:doaj.org-article:d3ec3990da20405cbf99c3d25883b6bd2021-12-02T05:03:21ZMultiple conformations in solution of the maize C4-phosphoenolpyruvate carboxylase isozyme2405-844010.1016/j.heliyon.2021.e08464https://doaj.org/article/d3ec3990da20405cbf99c3d25883b6bd2021-11-01T00:00:00Zhttp://www.sciencedirect.com/science/article/pii/S2405844021025676https://doaj.org/toc/2405-8440The photosynthetic phosphoenolpyruvate carboxylase isozyme from C4 plants (PEPC-C4) has a complex allosteric regulation, involving positive cooperativity in binding the substrate phosphoenolpyruvate as well as positive and negative allosteric effectors. Besides the proposed R- and T-states, previous kinetic results suggested functionally relevant different R-states of the maize enzyme (ZmPEPC-C4) elicited by PEP or its two kinds of activators, glucose 6-phosphate or glycine. To detect these different R-state conformations, we used as conformational probes the fluorescence of 8-anilino-1-naphthalene sulfonate (ANS), near-UV circular dichroism (CD) spectroscopy, and limited proteolysis by trypsin. Phosphoenolpyruvate and malate binding caused distinct concentration-dependent fluorescence changes of ZmPEPC-C4/ANS, suggesting that they elicited conformational states different from that of the free enzyme, while glucose 6-phosphate or glycine binding did not produce fluorescence changes. Differences were also observed in the near UV CD spectra of the enzyme, free or complexed with its substrate or allosteric effectors. Additionally, differences in the trypsin-digestion fragmentation patterns, as well as in the susceptibility of the free and complexed enzyme to digestion and digestion-provoked loss of activity, provided evidence of several ZmPEPC-C4 conformations in solution elicited by the substrate and the allosteric effectors. Using the already reported ZmPEPC-C4 crystal structures and bioinformatics methods, we predicted that the most probable trypsin-cleavage sites are located in superficial flexible regions, which seems relevant for the protein dynamics underlying the function and allosteric regulation of this enzyme. Together, our findings agree with previous kinetic results, shed light on this enzyme's complex allosteric regulation, and place ZmPEPC-C4 in the growing list of allosteric enzymes possessing an ensemble of closely related R-state conformations.Fátima Barreda-HuertaIsmael Bustos-JaimesCarlos Mújica-JiménezRosario A. Muñoz-ClaresElsevierarticleZea mays L.C4 metabolismAllosteric regulationLigand-induced conformational changesANS fluorescenceCD spectroscopyScience (General)Q1-390Social sciences (General)H1-99ENHeliyon, Vol 7, Iss 11, Pp e08464- (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Zea mays L. C4 metabolism Allosteric regulation Ligand-induced conformational changes ANS fluorescence CD spectroscopy Science (General) Q1-390 Social sciences (General) H1-99
spellingShingle	Zea mays L. C4 metabolism Allosteric regulation Ligand-induced conformational changes ANS fluorescence CD spectroscopy Science (General) Q1-390 Social sciences (General) H1-99 Fátima Barreda-Huerta Ismael Bustos-Jaimes Carlos Mújica-Jiménez Rosario A. Muñoz-Clares Multiple conformations in solution of the maize C4-phosphoenolpyruvate carboxylase isozyme
description	The photosynthetic phosphoenolpyruvate carboxylase isozyme from C4 plants (PEPC-C4) has a complex allosteric regulation, involving positive cooperativity in binding the substrate phosphoenolpyruvate as well as positive and negative allosteric effectors. Besides the proposed R- and T-states, previous kinetic results suggested functionally relevant different R-states of the maize enzyme (ZmPEPC-C4) elicited by PEP or its two kinds of activators, glucose 6-phosphate or glycine. To detect these different R-state conformations, we used as conformational probes the fluorescence of 8-anilino-1-naphthalene sulfonate (ANS), near-UV circular dichroism (CD) spectroscopy, and limited proteolysis by trypsin. Phosphoenolpyruvate and malate binding caused distinct concentration-dependent fluorescence changes of ZmPEPC-C4/ANS, suggesting that they elicited conformational states different from that of the free enzyme, while glucose 6-phosphate or glycine binding did not produce fluorescence changes. Differences were also observed in the near UV CD spectra of the enzyme, free or complexed with its substrate or allosteric effectors. Additionally, differences in the trypsin-digestion fragmentation patterns, as well as in the susceptibility of the free and complexed enzyme to digestion and digestion-provoked loss of activity, provided evidence of several ZmPEPC-C4 conformations in solution elicited by the substrate and the allosteric effectors. Using the already reported ZmPEPC-C4 crystal structures and bioinformatics methods, we predicted that the most probable trypsin-cleavage sites are located in superficial flexible regions, which seems relevant for the protein dynamics underlying the function and allosteric regulation of this enzyme. Together, our findings agree with previous kinetic results, shed light on this enzyme's complex allosteric regulation, and place ZmPEPC-C4 in the growing list of allosteric enzymes possessing an ensemble of closely related R-state conformations.
format	article
author	Fátima Barreda-Huerta Ismael Bustos-Jaimes Carlos Mújica-Jiménez Rosario A. Muñoz-Clares
author_facet	Fátima Barreda-Huerta Ismael Bustos-Jaimes Carlos Mújica-Jiménez Rosario A. Muñoz-Clares
author_sort	Fátima Barreda-Huerta
title	Multiple conformations in solution of the maize C4-phosphoenolpyruvate carboxylase isozyme
title_short	Multiple conformations in solution of the maize C4-phosphoenolpyruvate carboxylase isozyme
title_full	Multiple conformations in solution of the maize C4-phosphoenolpyruvate carboxylase isozyme
title_fullStr	Multiple conformations in solution of the maize C4-phosphoenolpyruvate carboxylase isozyme
title_full_unstemmed	Multiple conformations in solution of the maize C4-phosphoenolpyruvate carboxylase isozyme
title_sort	multiple conformations in solution of the maize c4-phosphoenolpyruvate carboxylase isozyme
publisher	Elsevier
publishDate	2021
url	https://doaj.org/article/d3ec3990da20405cbf99c3d25883b6bd
work_keys_str_mv	AT fatimabarredahuerta multipleconformationsinsolutionofthemaizec4phosphoenolpyruvatecarboxylaseisozyme AT ismaelbustosjaimes multipleconformationsinsolutionofthemaizec4phosphoenolpyruvatecarboxylaseisozyme AT carlosmujicajimenez multipleconformationsinsolutionofthemaizec4phosphoenolpyruvatecarboxylaseisozyme AT rosarioamunozclares multipleconformationsinsolutionofthemaizec4phosphoenolpyruvatecarboxylaseisozyme
_version_	1718400739652403200

Multiple conformations in solution of the maize C4-phosphoenolpyruvate carboxylase isozyme

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