Structural insights into the allosteric site of Arabidopsis NADP-malic enzyme 2: role of the second sphere residues in the regulatory ... View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2021-07-31

AUTHORS

Mariel Claudia Gerrard Wheeler, Cintia Lucía Arias, Juliana da Fonseca Rezende e Mello, Nuria Cirauqui Diaz, Carlos Rangel Rodrigues, María Fabiana Drincovich, Alessandra Mendonça Teles de Souza, Clarisa Ester Alvarez

ABSTRACT

Key messageNADP-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.AbstractStructure–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.Graphic abstract More... »

PAGES

37-48

Identifiers

URI

http://scigraph.springernature.com/pub.10.1007/s11103-021-01176-2

DOI

http://dx.doi.org/10.1007/s11103-021-01176-2

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1140103993

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/34333694


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78 modeling
79 modeling approach
80 modification
81 molecular docking
82 molecular modeling approach
83 mutagenesis
84 new insights
85 normal mode analysis
86 number
87 optimization
88 overall functioning
89 particular interest
90 pocket
91 primary structure
92 reduction
93 region
94 regulation
95 residues
96 results
97 role
98 second sphere
99 second-sphere residues
100 sequence
101 signal transmission
102 site model
103 site-directed mutagenesis
104 sites
105 small modifications
106 species
107 sphere
108 sphere residues
109 structural determinants
110 structural insights
111 structural modeling
112 structure
113 study
114 substitution
115 substrate
116 substrate affinity
117 thaliana
118 transmission
119 tryptophan
120 understanding
121 variety
122 wide variety
123 work
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