Crystallization and preliminary crystallographic analysis of gallate dioxygenase DesB from Sphingobium sp. SYK-6

Abstract

Gallate dioxygenase (DesB) from Sphingobium sp. SYK-6, which belongs to the type II extradiol dioxygenase family, was purified and crystallized using the hanging-drop vapour-diffusion method. Two crystal forms were obtained. The form I crystal belonged to space group C2, with unit-cell parameters a = 136.2, b = 53.6, c = 55.1 angstrom, beta = 112.8 degrees, and diffracted to 1.6 angstrom resolution. The form II crystal belonged to space group P2(1), with unit-cell parameters a = 56.2, b = 64.7, c = 116.1 angstrom, beta = 95.1 degrees, and diffracted to 1.9 angstrom resolution. A molecular-replacement calculation using LigAB as a search model yielded a satisfactory solution for both crystal forms.

Figure 1

(a) Degradation pathway of syringate and vanillate in Sphingobium sp. SYK-6. DesA, syringate O-demethylase; LigM, vanillate/3MGA O-demethylase; DesB, gallate dioxygenase; LigAB, PCA 4,5-dioxygenase; DesZ, 3MGA 3,4-dioxygenase; LigI, PDC hydrolase; LigC, 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase; 3MGA, 3-O-methylgallate; OMA, 4-oxalomesaconate; PCA, protocatechuate; CHMOD, 4-carboxy-2-hydroxy-6-methoxy-6-oxohexa-2,4-dienoate; PDC, 2-pyrone-4,6-dicarboxylate; CHMS, 4-carboxy-2-hydroxymuconate-6-semialdehyde. (b) Schematic drawing of LigAB, DesB and DesZ from Sphingobium sp. SYK-6.

Figure 2

Crystals of DesB. The largest dimension of the crystal is approximately 300 µm.

Figure 3

X-ray diffraction pattern of the DesB crystal. The diffraction data were collected on BL-5A of PF using an ADSC Quantum 315r CCD detector.