Crystallization and preliminary X-ray analysis of 4-pyridoxolactonase fromMesorhizobium loti

Abstract

4-Pyridoxolactonase from Mesorhizobium loti MAFF303099 has been overexpressed in Escherichia coli. The recombinant enzyme was purified and was crystallized by the sitting-drop vapour-diffusion method using PEG 4000 and ammonium sulfate as precipitants. Crystals of the free enzyme (form I) and of the 5-pyridoxolactone-bound enzyme (form II) grew under these conditions. Crystals of form I diffracted to 2.0 Å resolution and belonged to the monoclinic space group C2, with unit-cell parameters a = 77.93, b = 38.88, c = 81.60 Å , = 117.33 . Crystals of form II diffracted to 1.9 Å resolution and belonged to the monoclinic space group C2, with unit-cell parameters a = 86.24, b = 39.35, c = 82.68 Å , = 118.02. The calculated V M values suggested that the asymmetric unit contains one molecule in both crystal forms.

Structure of 4-pyridoxolactonase fromMesorhizobium loti

“…The purified native-His 6 and SeMet-His 6 enzymes were concentrated to 8 mg ml À1 by ultrafiltration using a Vivaspin 20. The native enzyme without the His 6 tag has already been crystallized using reservoir solution consisting of 30%(w/v) PEG 4000, 200 mM ammonium acetate, 100 mM sodium acetate (Matsuda et al, 2009). After optimization, both the native-His 6 and SeMet-His 6 enzymes were crystallized by mixing equal volumes (2 ml:2 ml) of enzyme solution (8 mg ml À1 in crystallization buffer) and reservoir solution [30%(w/v) PEG 4000, 150 mM ammonium acetate, 100 mM sodium acetate] by the sitting-drop method for one month at 277 K. The native-His 6 -5PAL complex was not crystallized under this condition.…”

“…A preliminary X-ray crystallographic analysis of 4-pyridoxolactonase from M. loti has been reported (Matsuda et al, 2009). However, the crystal structure has not been elucidated owing to the phase problem.…”