Abstract
Halophilic enzymes need high salt concentrations for activity and stability and are considered a promising source for biotechnological applications. The model study for haloadaptation has been proteins from the Halobacteria class of Archaea, where common structural characteristics have been found. However, the effect of salt on enzyme function and conformational dynamics has been much less explored. Here we report the structural and kinetic characteristics of glucose-6-phosphate dehydrogenase from Haloferax volcanii (HvG6PDH) belonging to the short-chain dehydrogenases/reductases (SDR) superfamily. The enzyme was expressed in Escherichia coli and successfully solubilized and refolded from inclusion bodies. The enzyme is active in the presence of several salts, though the maximum activity is achieved in the presence of KCl, mainly by an increment in the kcat value, that correlates with a diminution of its flexibility according to molecular dynamics simulations. The high KM for glucose-6-phosphate and its promiscuous activity for glucose restrict the use of HvG6PDH as an auxiliary enzyme for the determination of halophilic glucokinase activity. Phylogenetic analysis indicates that SDR-G6PDH enzymes are exclusively present in Halobacteria, with HvG6PDH being the only enzyme characterized. Homology modeling and molecular dynamics simulations of HvG6PDH identified a conserved NLTX2H motif involved in glucose-6-phosphate interaction at high salt concentrations, whose residues could be crucial for substrate specificity. Structural differences in its conformational dynamics, potentially related to the haloadaptation strategy, were also determined.
Highlights
The oxidation of glucose-6-phosphate (G6P) to 6-phosphoglucono-1,5-lactone plays an essential role in the oxidative metabolism of glucose, being the first step of the oxidative branch of the pentose phosphate pathway (Fraenkel and Vinopal, 1973)
We identified putative residues involved in G6P specificity, and by molecular dynamics simulations in the presence and in the absence of salt, we identified structural differences in its conformational dynamics potentially related with the haloadaptation strategy
This was the case for the expression of HvG6PDH in E. coli, where we used a solubilization and refolding protocol, similar to the one described by Pire (Pire et al, 2001), that enabled us to obtain the purified enzyme in a soluble and active form, with kinetic parameters comparable to those reported by Pickl and Schönheit (2015), who characterized the enzyme obtained from the original organism (H. volcanii)
Summary
The oxidation of glucose-6-phosphate (G6P) to 6-phosphoglucono-1,5-lactone plays an essential role in the oxidative metabolism of glucose, being the first step of the oxidative branch of the pentose phosphate pathway (Fraenkel and Vinopal, 1973). The first type corresponds to the well-known G6PDH that belongs to the G6PDH family (Csonka and Fraenkel, 1977; Stincone et al, 2014) These enzymes are tetrameric, with an N-terminal Rossman fold involved in NADP+ or NAD+ binding and a C-terminal α/β domain where G6P binds (Rowland et al, 1994). A new type of G6PDH belonging to the short-chain dehydrogenases/reductases (SDR) superfamily has been reported in Halobacteria (Pickl and Schönheit, 2015) The enzymes from this superfamily are mainly homodimers that shared a Rossmann-fold of a parallel β-sheet of six to seven β-strands flanked by three to four α-helices from both sides (Kallberg et al, 2010) and include different NAD(P)(H)-dependent oxidoreductases (Kavanagh et al, 2008)
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