Cloning, expression, purification, crystallization and preliminary crystallographic studies of UgdG, an UDP-glucose dehydrogenase from Sphingomonas elodea ATCC 31461

Abstract

Gellan gum, a commercial gelling agent produced by Sphingomonas elodea ATCC 31461, is a high-value microbial exopolysaccharide. UDP-glucose dehydrogenase (UGD; EC 1.1.1.22) is responsible for the NAD-dependent twofold oxidation of UDP-glucose to UDP-glucuronic acid, one of the key components for gellan biosynthesis. S. elodea ATCC 31461 UGD, termed UgdG, was cloned, expressed, purified and crystallized in native and SeMet-derivatized forms in hexagonal and tetragonal space groups, respectively; the crystals diffracted X-rays to 2.40 and 3.40 A resolution, respectively. Experimental phases were obtained for the tetragonal SeMet-derivatized crystal form by a single-wavelength anomalous dispersion experiment. This structure was successfully used as a molecular-replacement probe for the hexagonal crystal form of the native protein.

Figure 1

Crystals of (a) native UgdG in a hexagonal space group (approximate dimensions of 0.04, 0.04, 0.06 mm) and (b) SeMet-derivative UgdG in a tetragonal space group (approximate dimensions of 0.05, 0.05, 0.08 mm).

Figure 2

Electron-density maps showing a helical motif section (bonds shown as sticks, with carbon in yellow, nitrogen in blue and oxygen in red) of S. elodea UgdG (a) from the experimental SeMet-derivative σ_A map (Read, 1986 ▶) obtained by RESOLVE (Terwilliger, 2004 ▶) after twofold NCS averaging at 3.4 Å resolution and contoured at 2σ (dark blue mesh) and (b) from the molecular-replacement solution obtained by Phaser (McCoy et al., 2007 ▶): a σ_A OMIT map (Read, 1986 ▶) at 2.4 Å resolution contoured at 1.5σ (dark blue mesh), where four residues of the helical motif were omitted from the model in the map calculation using REFMAC5 (Murshudov et al., 1997 ▶).