A novel bacterial class V dye-decolourizing peroxidase from the extremophile Deinococcus radiodurans: cloning, expression optimization, purification, crystallization, initial characterization and X-ray diffraction analysis

摘要

Deinococcus radiodurans is a bacterium with extreme resistance to desiccation and radiation. The resistance mechanism is unknown, but an efficient reactive oxygen species (ROS) scavenging system and DNA-repair and DNA-protection mechanisms are believed to play important roles. Here, the cloning and smalland medium-scale expression tests of a novel dye-decolourizing peroxidase from D. radiodurans (DrDyP) using three different Escherichia coli strains and three different temperatures in order to identify the optimum conditions for the expression of recombinant DrDyP are presented. The best expression conditions were used for large-scale expression and yielded ~10 mg recombinant DrDyP per litre of culture after purification. Initial characterization experiments demonstrated unusual features with regard to the haem spin state, which motivated the crystallization experiment. The obtained crystals were used for data collection and diffracted to 2.2 ? resolution. The crystals belonged to the trigonal space group P3_1 or P3_2, with unit-cell parameters a = b = 64.13, c = 111.32 ? , and are predicted to contain one DrDyP molecule per asymmetric unit. Structure determination by molecular replacement using previously determined structures of dye-decolourizing peroxidases with ~30% sequence identity at ~2 A° resolution as templates are ongoing.