Crystallization of the extracellular rubber oxygenase RoxA fromXanthomonassp. strain 35Y

Abstract

Rubber oxygenase A (RoxA) from Xanthomonas sp. strain 35Y is an extracellular dioxygenase that is capable of cleaving the double bonds of poly(cis-1,4-isoprene) into short-chain isoprene units with 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD) as the major cleavage product. Crystals of the dihaem c-type cytochrome RoxA were grown by sitting-drop vapour diffusion using polyethylene glycol as a precipitant. RoxA crystallized in space group P2 1 , with unit-cell parameters a = 72.4, b = 97.1, c = 101.1 Å , = 98.39 , resulting in two monomers per asymmetric unit. Diffraction data were collected to a limiting resolution of 1.8 Å . Despite a protein weight of 74.1 kDa and only two iron sites per monomer, phasing was successfully carried out by multiple-wavelength anomalous dispersion.

Structure of the processive rubber oxygenase RoxA from Xanthomonas sp

“…The four Fe sites were located with SHELXD (41). SHARP (42) was used for site refinement and phase calculations (15). The quality of the resulting electron density map was sufficient for model building using Coot (43), and a nearly complete model was built manually before crystallographic refinement was carried out with REFMAC (44).…”

“…Crystals of RoxA were obtained by the sitting drop vapor diffusion method as described previously (15). A fourwavelength anomalous dispersion experiment was carried out at the K-edge of iron to optimally exploit the phasing power of the two metal ions in the heme groups of RoxA (15).…”

“…Crystals of RoxA were obtained by the sitting drop vapor diffusion method as described previously (15). A fourwavelength anomalous dispersion experiment was carried out at the K-edge of iron to optimally exploit the phasing power of the two metal ions in the heme groups of RoxA (15). In addition, a high-resolution dataset was obtained from crystals of oxidized RoxA that yielded diffraction data to resolutions better than 1.8 Å. Crystals of native RoxA belong to the monoclinic space group P2 1 , with two monomers per asymmetric unit.…”

Functional Identification of Rubber Oxygenase (RoxA) in Soil and Marine Myxobacteria

“…Determination of the recently solved three-dimensional structure of the RoxA Xsp protein showed that the N-terminal heme has only one axial amino acid ligand (H 195 ) on the proximal side and that dioxygen represents the distal heme ligand (18,19). The N-terminal heme constitutes the active site of RoxA.…”