Crystallization and preliminary X-ray analysis of 4-pyridoxolactonase from Mesorhizobium 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 A resolution and belonged to the monoclinic space group C2, with unit-cell parameters a = 77.93, b = 38.88, c = 81.60 A, beta = 117.33 degrees. Crystals of form II diffracted to 1.9 A resolution and belonged to the monoclinic space group C2, with unit-cell parameters a = 86.24, b = 39.35, c = 82.68 A, beta = 118.02 degrees. The calculated V(M) values suggested that the asymmetric unit contains one molecule in both crystal forms.

Figure 1

Reactions catalyzed by 4-pyridoxolactonase. (a) Hydrolysis of 4-pyridoxolactone to 4-pyridoxic acid. (b) Hydrolysis of N-hexanoyl-d,l-homoserine lactone to N-­hexanoyl-d,l-homoserine.

Figure 2

SDS–PAGE analysis of purified 4-pyridoxolactonase. Lane 1, molecular markers (kDa). Lane 2, purified 4-pyridoxolactonase for crystallization.

Figure 3

Crystals of 4-pyridoxolactonase. (a) Crystal of form I. (b) Crystal of form II, in which the enzyme binds 5-pyridoxolactone. The scale bars are 0.1 mm in length.

Figure 4

Diffraction images of M. loti 4-pyridoxolactonase. (a) Form I crystal with a resolution scale of 1.9 Å indicated by the ring. (b) Form II crystal with a resolution scale of 1.9 Å indicated by the ring.