Crystallization and preliminary X-ray analysis of pyridoxine 4-oxidase, the first enzyme in pyridoxine degradation pathway I

Abstract

Vitamin B(6)-degradation pathway I has recently been identified in Mesorhizobium loti MAFF303099. Pyridoxine 4-oxidase, an FAD-dependent enzyme, is the first enzyme in this pathway and catalyzes the irreversible oxidation of pyridoxine to pyridoxal. The enzyme was overexpressed in Escherichia coli with a His(6) tag and purified. The recombinant enzyme was crystallized at 277 K by the sitting-drop vapour-diffusion method using PEG 4000 as the precipitant. The crystal, which belonged to space group P2(1)2(1)2(1) with unit-cell parameters a = 62.38, b = 79.44, c = 136.43 Å, diffracted to 2.2 Å resolution. The calculated V(M) value (3.19 Å(3) Da(-1)) suggested that the asymmetric unit contained one molecule.

Figure 1

The reaction catalyzed by pyridoxine 4-oxidase.

Figure 2

SDS–PAGE analysis of the purified pyridoxine 4-oxidase. Lane 1, molecular-weight markers (labelled in kDa). Lane 2, purified enzyme used for crystallization (4 µg).

Figure 3

Crystal of pyridoxine 4-oxidase. The scale bar is 0.2 mm in length.

Figure 4

Diffraction image of pyridoxine 4-oxidase. The ring indicates a resolution of 2.2 Å.