Characterization of Escherichia coli OtsA, a trehalose-6-phosphate synthase from glycosyltransferase family 20

The Ots gene cluster of Escherichia coli encodes the synthetic apparatus for the formation of α,α-1,1-trehalose, a non-reducing glucose disaccharide. The otsA gene encodes a trehalose-6-phosphate synthase, a glycosyltransferase which catalyses the synthesis of α,α-­1,1-trehalose-6-phosphate from glucose-6-phosphate using a UDP-glucose donor. It has been classified into glycosyltransferase family GT-20 based upon amino-acid sequence similarities. The otsA gene has been cloned and recombinant protein overexpressed using a pET-based system in E. coli BL21 cells. The recombinant protein (MW ≃ 54.7 kDa) is active and has been crystallized in two forms suitable for X-ray diffraction analysis. The first is orthorhombic, P2₁2₁2₁, with unit-cell parameters a = 104.1, b = 127.8, c = 179.9 Å. Data for this form have been collected to 3.0 Å resolution at the CLRC Daresbury Synchrotron Radiation Source. The second form has unit-cell parameters a = b = 141.9, c = 317.8 Å and displays the apparent space group P4₂. These crystals diffract beyond 2 Å resolution, but display merohedral twinning.