Structural and functional insights into the flexible β‐hairpin of glycerol dehydrogenase

Abstract

During glycerol metabolism, the initial step of glycerol oxidation is catalysed by glycerol dehydrogenase (GDH), which converts glycerol to dihydroxyacetone in a NAD+‐dependent manner via an ordered Bi‐Bi kinetic mechanism. Structural studies conducted with GDH from various species have mainly elucidated structural details of the active site and ligand binding. However, the structure of the full GDH complex with both cofactor and substrate bound is not determined, and thus, the structural basis of the kinetic mechanism of GDH remains unclear. Here, we report the crystal structures of Escherichia coli GDH with a substrate analogue bound in the absence or presence of NAD+. Structural analyses including molecular dynamics simulations revealed that GDH possesses a flexible β‐hairpin, and that during the ordered progression of the kinetic mechanism, the flexibility of the β‐hairpin is reduced after NAD+ binding. It was also observed that this alterable flexibility of the β‐hairpin contributes to the cofactor binding and possibly to the catalytic efficiency of GDH. These findings suggest the importance of the flexible β‐hairpin to GDH enzymatic activity and shed new light on the kinetic mechanism of GDH.

Structural insights into the octamerization of glycerol dehydrogenase

“…Our previous structural study of GDH complexed with NAD + and an inhibitor, tris(hydroxymethyl)aminomethane (Tris), provided insight into the GDH ternary complex [ 23 ]. However, due to the divergent chemical properties of Tris compared to glycerol, it remained uncertain whether the ternary complex elucidated was the same as that with the actual substrate bound.…”

“…Prior to crystallization, ZnCl 2 and NAD + were added to the purified GDH sample to final concentrations of 1 mM and 2 mM, respectively. The GDH NAD∙Gly crystal was prepared using methods similar to those previously reported [ 23 ]. The refined GDH NAD∙Gly crystal was obtained using the hanging-drop vapor diffusion method with a 1:1 mixture of protein solution and well solution containing 100 mM sodium phosphate dibasic and 100 mM citric acid (pH 4.2 adjusted with NaOH), 200 mM NaCl, and 10% ( w / v ) PEG 3000 at 20°C.…”

“…For simulations, models of GDH in three ligand-bound states were prepared using methods similar to those previously reported [ 23 ], but with molecules forming an octamer according to crystallographic symmetry. The simulations were carried out using the GROMACS version 2021.3 software package [ 29 ], with the Amber99SB-ILDN force field [ 30 ] for the protein and the ff14SB force field [ 31 ] for the ligands.…”

“…The N-domain includes six parallel β-sheets and ɑ-helices in a Rossmann fold arrangement, while the C-domain includes several ɑ-helices forming bundles [ 17 , 19 ]. An intriguing structural feature of GDH is the β-hairpin (β7–8) located between the two domains, which exhibits marked dynamic conformational variation depending on the presence of NAD + [ 23 ]. The flexibility of this β-hairpin was revealed to contribute to the enzyme’s efficiency.…”

“…Another noteworthy characteristic is the homo-octameric configuration observed for all reported GDH structures, with the exception of the enzyme from the evolutionarily distant Clostridium acetobutylicum ( C . acetobutylicum ) [ 17 – 23 ]. This structural attribute has also been observed in solution-based assays with various species, including E .…”