Computational modeling of the molecular basis for the calcium-dependence of the mannuronan C-5 epimerase AvAlgE6 from Azotobacter vinelandii

Margrethe Gaardløs; Anders Lervik; Sergey A Samsonov

doi:10.1016/j.csbj.2023.03.021

Computational modeling of the molecular basis for the calcium-dependence of the mannuronan C-5 epimerase AvAlgE6 from Azotobacter vinelandii

Comput Struct Biotechnol J. 2023 Mar 15:21:2188-2196. doi: 10.1016/j.csbj.2023.03.021. eCollection 2023.

Authors

Margrethe Gaardløs^{1

2}, Anders Lervik¹, Sergey A Samsonov²

Affiliations

¹ Faculty of Chemistry, University of Gdańsk, ul. Wita Stwosza 63, 80-308 Gdańsk, Poland.
² Department of Chemistry, NTNU Norwegian University of Science and Technology, N-7491 Trondheim, Norway.

Abstract

The mannuronan C-5 epimerases catalyze epimerization of β-d-mannuronic acid to α-l-guluronic acid in alginate polymers. The seven extracellular Azotobacter vinelandii epimerases (AvAlgE1-7) are calcium-dependent, and calcium is essential for the structural integrity of their carbohydrate binding R-modules. Ca²⁺ is also found in the crystal structures of the A-modules, where it is suggested to play a structural role. In this study, the structure of the catalytic A-module of the A. vinelandii mannuronan C-5 epimerase AvAlgE6 is used to investigate the role of this Ca²⁺. Molecular dynamics (MD) simulations with and without calcium reveal the possible importance of the bound Ca²⁺ in the hydrophobic packing of β-sheets. In addition, a putative calcium binding site is found in the active site, indicating a potential direct role of this calcium in the catalysis. According to the literature, two of the residues coordinating calcium in this site are essential for the activity. MD simulations of the interaction with bound substrate indicate that the presence of a calcium ion in this binding site increases the binding strength. Further, explicit calculations of the substrate dissociation pathways with umbrella sampling simulations show and energetically higher dissociation barrier when calcium is present. The present study eludes to a putative catalytic role of calcium in the charge neutralizing first step of the enzymatic reaction. In addition to the importance for understanding these enzymes' molecular mechanisms, this could have implications for engineering strategies of the epimerases in industrial alginate processing.

Keywords: Alginate; Calcium dependence; Mannuronan C-5 epimerases; Molecular dynamics; Reaction mechanism; Umbrella sampling.