Crystal Structure, SAXS and Kinetic Mechanism of Hyperthermophilic ADP-Dependent Glucokinase from Thermococcus litoralis Reveal a Conserved Mechanism for Catalysis

Jaime Andrés Rivas-Pardo,Victoria Guixé,Francisco J Fernandez,M Cristina Vega,Alejandra Herrera-Morande,Victor Castro-Fernandez,Beata G Vertessy

doi:10.1371/journal.pone.0066687

Jaime Andrés Rivas-Pardo, Victoria Guixé + Show 5 more

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https://doi.org/10.1371/journal.pone.0066687

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Abstract

ADP-dependent glucokinases represent a unique family of kinases that belong to the ribokinase superfamily, being present mainly in hyperthermophilic archaea. For these enzymes there is no agreement about the magnitude of the structural transitions associated with ligand binding and whether they are meaningful to the function of the enzyme. We used the ADP-dependent glucokinase from Termococcus litoralis as a model to investigate the conformational changes observed in X-ray crystallographic structures upon substrate binding and to compare them with those determined in solution in order to understand their interplay with the glucokinase function. Initial velocity studies indicate that catalysis follows a sequential ordered mechanism that correlates with the structural transitions experienced by the enzyme in solution and in the crystal state. The combined data allowed us to resolve the open-closed conformational transition that accounts for the complete reaction cycle and to identify the corresponding clusters of aminoacids residues responsible for it. These results provide molecular bases for a general mechanism conserved across the ADP-dependent kinase family.

Highlights

The Embden–Meyerhof pathway is the most common route for the degradation of glucose
Ray crystallographic data provide important new perspectives on the conformational changes and dynamics of TlGK during catalysis, which are relevant for other ADP-dependent hyperthermophilic kinases
When the enzyme is in ligand-free state, the large and small domains are distant from each other and there are no discernible interactions between residues from either domain

Summary

Introduction

The Embden–Meyerhof pathway is the most common route for the degradation of glucose This metabolic pathway is generally highly conserved between different organisms, several archaea of the Euryarchaeota have evolved major modifications with only four of the classical enzymes present in the canonical pathway. Some authors have attributed the presence of ADP-dependent enzymes to a matter of metabolic adaptation to high temperatures and to starvation conditions, several facts indicate that the presence of these proteins in the central metabolism of archaea is not related to the hyperthermophilic life style [3] Even though these ADP-dependent kinases show no sequence similarity to ATP-dependent enzymes known to date, the determination of their three dimensional structures allowed their reliable classification as members of the ribokinase superfamily. The small domain, which could function as an active-site lid to protect substrates from hydrolysis, has been proposed as a phylogenetic marker for the evolution of this superfamily [4]

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