Purification, crystallization and preliminary X-ray analysis of L-sorbose reductase from Gluconobacter frateurii complexed with L-sorbose or NADPH

Abstract

NADPH-dependent L-sorbose reductase (SR) from Gluconobacter frateurii was expressed in Escherichia coli, purified and crystallized with L-sorbose or NADPH using the sitting-drop vapour-diffusion method at 293 K. Crystals of the SR-L-sorbose complex and the SR-NADPH complex were obtained using reservoir solutions containing PEG 2000 or PEG 400 as precipitants and diffracted X-rays to 2.38 and 1.90 A resolution, respectively. The crystal of the SR-L-sorbose complex belonged to space group C222(1), with unit-cell parameters a = 124.2, b = 124.1, c = 60.8 A. The crystal of the SR-NADPH complex belonged to space group P2(1), with unit-cell parameters a = 124.3, b = 61.0, c = 124.5 A, beta = 89.99 degrees . The crystals contained two and eight molecules, respectively, in the asymmetric unit.

Figure 1

Crystals of SR complexed with (a) l-sorbose and (b) NADPH. The scale bars are 100 µm in length.

Figure 2

X-ray diffraction images of (a) the SR–l-sorbose complex crystal and (b) the SR–NADPH complex crystal. The circles display resolutions of (a) 2.38 Å and (b) 1.90 Å.