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.
Glycerol dehydrogenase (GldA) from Escherichia coli is a Zn2+‐containing alcohol dehydrogenase which catalyzes the NAD+‐dependent oxidation of glycerol to dihydroxyacetone. In this study, GldA has been cloned, over‐expressed, and isolated by an affinity and an ion‐exchange chromatography. GldA shows a strong intrinsic fluorescence at 320 nm, when excited at 280 nm. The fluorescence intensity decreases in the presence of NAD+, NADH, and dihydroxyacetone, the substrate and products for GldA, which allows us to determine the dissociation constants for those molecules as 110.6 ± 5.0 μM, 9,1 ± 0.6 μM, 33.3 ± 2.3 mM, respectively. The dissociation constant for NAD+ was similar to the kinetic constant, KM. Guanosine‐5′‐diphosphate 3′‐diphosphate (ppGpp), accumulated in E. coli when starved for amino acids, nutrients, and phosphate, serves as a global regulator in replication, transcription, and translation. In this study, the fluorescence intensity of GldA also decreases in the presence of ppGpp and the dissociation constant for ppGpp is calculated as 108.9 ± 8.6 μM. ppGpp increases GldA activity with the half maximal activation at 33.1 ± 3.1 μM. On the contrary, GTP and GDP inhibit GldA, with the inhibition constants of 16.1 ± 1.1 mM and 10.6 ± 0.3 mM, respectively. Tris(hydroxymethyl)aminomethane serves as a competitive inhibitor against glycerol. GTP and GDP also bind to GldA with the dissociation constants of 60.0 ± 0.8 and 61.0 ± 1.3 μM, respectively. These results suggest that GTP and GDP bind to GldA as strongly as ppGpp but only ppGpp activates GldA. This study shows that ppGpp binds to GldA and activates its activity for the first time. It is also suggested that the strong intrinsic fluorescence of enzymes and their changes in the presence of various ligands can be utilized to measure the binding affinities for those ligands. The method described here is especially effective for bindings with the relatively lower affinities.
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