Purification, crystallization and preliminary X-ray diffraction analysis of a novel keto-deoxy-D-galactarate (KDG) dehydratase from Agrobacterium tumefaciens

Abstract

D-galacturonic acid is the main component of pectin. It could be used to produce affordable renewable fuels, chemicals and materials through biotechnical conversion. Keto-deoxy-D-galactarate (KDG) dehydratase is an enzyme in the oxidative pathway of D-galacturonic acid in Agrobacterium tumefaciens (At). It converts 3-deoxy-2-keto-L-threo-hexarate to α-ketoglutaric semialdehyde. At KDG dehydratase was crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 169.1, b = 117.8, c = 74.3 Å, β = 112.4° and an asymmetric unit of four monomers. X-ray diffraction data were collected to 1.9 Å resolution using synchrotron radiation. The three-dimensional structure of At KDG dehydratase will provide valuable information on the function of the enzyme and will allow it to be engineered for biorefinery-based applications.

Keywords: Agrobacterium tumefaciens; d-galacturonic acid; keto-deoxy-d-galactarate dehydratase; oxidative pathway.

Figure 1

SDS–PAGE analysis of purified At KDG dehydratase. Lane M contains molecular-weight markers (labelled in kDa), lane 1 contains purified At KDG dehydratase and lane 2 contains a crude E. coli cell extract of the At KDG dehydratase-producing strain. The proteins were visualized using the 10% Criterion Stain-Free gel-imaging system (Bio-Rad).

Figure 2

At KDG dehydratase crystals (a) from the initial screening and (b) after optimizing the crystallization conditions. The optimized crystallization conditions consisted of 0.1 M Bicine pH 8.5, 0.2 M sodium formate, 15%(w/v) PEG MME 5000 and the crystals grew in 3–4 d.