Expression, purification, crystallization and initial crystallographic characterization of the p-hydroxybenzoate hydroxylase from Corynebacterium glutamicum

Abstract

p-Hydroxybenzoate hydroxylase (PHBH) is an FAD-dependent monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate (pOHB) to 3,4-dihydroxybenzoate in an NADPH-dependent reaction and plays an important role in the biodegradation of aromatic compounds. PHBH from Corynebacterium glutamicum was crystallized using the hanging-drop vapour-diffusion method in the presence of NaH(2)PO(4) and K(2)HPO(4) as precipitants. X-ray diffraction data were collected to a maximum resolution of 2.5 A on a synchrotron beamline. The crystal belongs to the hexagonal space group P6(3)22, with unit-cell parameters a = b = 94.72, c = 359.68 A, gamma = 120 degrees . The asymmetric unit contains two molecules, corresponding to a packing density of 2.65 A(3) Da(-1). The structure was solved by molecular replacement. Structure refinement is in progress.

Figure 1

Enzyme reaction catalyzed by p-hydroxybenzoate hydroxylase. The reaction involves FAD-dependent oxygenation of the aromatic compound coupled to oxidation of NADPH.

Figure 2

Hexagonal crystal of CgPHBH. The yellow colour of the crystal indicates that the protein contains FAD. The crystals have approximate dimensions of 300 × 150 × 100 µm.