Mannuronan C-5 epimerases (ManC5-Es) are produced by brown algae and some bacteria, such as Azotobacter and some Pseudomonas species. It can convert the transformation of β-D-mannuronic acid (M) to α-L-guluronic acid (G) in alginate with different patterns of epimerization. Alginate with different compositions and monomer sequences possess different properties and functions, which have been utilized in industries for various purposes. Therefore, ManC5-Es are key enzymes that are involved in the modifications of alginate for fuel, chemical, and industrial applications. Focusing on ManC5-Es, this review introduces and summarizes the methods of ManC5-Es activity assay especially the most widely used nuclear magnetic resonance spectroscopy method, characterization of the ManC5-Es from different origins especially the research progress of its enzymatic properties and product block distributions, and the catalytic mechanism of ManC5-E based on the resolved enzyme structures. Additionally, some potential future research directions are also outlooked.

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Mannuronan C-5 Epimerases: Review of Activity Assays, Enzyme Characteristics, Structure, and Mechanism

The adaptability of the active site to amplify the secondary function is supposed to be the fundamental cause of the promiscuity and the evolution of new functions in the enzymes. In most cases, mutations occur close to the active site and/or in the catalytic site to change the active site plasticity to accommodate the non-native substrate. In the present study, using MD simulations and hybrid QM/MM calculations, we have shown a way to enhance the promiscuity, i.e., the allostery-driven promiscuity. Using a case study of the AEE enzyme where the capping loop recognizes the substrate, herein, we show that a single site mutation (D321G) far from the capping loop can induce a large conformational change in the capping loop to recognize different substrates for different functions. The QM/MM calculations for the WT and mutated enzyme provide a first validation of the mechanism of 1,1-proton transfer and dehydration by the AEE enzyme. Since AEE epimerase possesses a highly conserved TIM-barrel fold, we believe that our study provides a crucial lead to understanding the mechanism of emergence of secondary function which can be useful to repurpose ancient enzymes for modern usage. 

/pdf/combined-md-and-qm-mm-calculations-reveal-allostery-driven-2i6snrcv.pdf

08065912405

Papers

Combined MD and QM/MM Calculations Reveal Allostery‐Driven Promiscuity in Dipeptide Epimerases of Enolase Family