Structure of glycerol dehydrogenase (GldA) from Escherichia coli.

Abstract

Escherichia coli (strain K-12, substrain MG1655) glycerol dehydrogenase (GldA) is required to catalyze the first step in fermentative glycerol metabolism. The protein was expressed and purified to homogeneity using a simple combination of heat-shock and chromatographic methods. The high yield of the protein (∼250 mg per litre of culture) allows large-scale production for potential industrial applications. Purified GldA exhibited a homogeneous tetrameric state (∼161 kDa) in solution and relatively high thermostability (Tm = 65.6°C). Sitting-drop sparse-matrix screens were used for protein crystallization. An optimized condition with ammonium sulfate (2 M) provided crystals suitable for diffraction, and a binary structure containing glycerol in the active site was solved at 2.8 Å resolution. Each GldA monomer consists of nine β-strands, thirteen α-helices, two 310-helices and several loops organized into two domains, the N- and C-terminal domains; the active site is located in a deep cleft between the two domains. The N-terminal domain contains a classic Rossmann fold for NAD+ binding. The O1 and O2 atoms of glycerol serve as ligands for the tetrahedrally coordinated Zn2+ ion. The orientation of the glycerol within the active site is mainly stabilized by van der Waals and electrostatic interactions with the benzyl ring of Phe245. Computer modeling suggests that the glycerol molecule is sandwiched by the Zn2+ and NAD+ ions. Based on this, the mechanism for the relaxed substrate specificity of this enzyme is also discussed.