Open AccessCloning, expression, purification, crystallization and preliminary X‐ray diffraction analysis of chlorite dismutase: a detoxifying enzyme producing molecular oxygen
Author(s) -
De Geus Daniël C.,
Thomassen Ellen A. J.,
Van Der Feltz Clarisse L.,
Abrahams Jan Pieter
Publication year - 2008
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309108020551
Subject(s) - chlorite , chemistry , chlorate , superoxide dismutase , enzyme , dismutase , escherichia coli , crystallization , nuclear chemistry , biochemistry , inorganic chemistry , biology , organic chemistry , gene , paleontology , quartz
Chlorite dismutase, a homotetrameric haem‐based protein, is one of the key enzymes of (per)chlorate‐reducing bacteria. It is highly active (>2 kU mg −1 ) in reducing the toxic compound chlorite to the innocuous chloride anion and molecular oxygen. Chlorite itself is produced as the intermediate product of (per)chlorate reduction. The chlorite dismutase gene in Azospira oryzae strain GR‐1 employing degenerate primers has been identified and the active enzyme was subsequently overexpressed in Escherichia coli . Chlorite dismutase was purified, proven to be active and crystallized using sitting drops with PEG 2000 MME, KSCN and ammonium sulfate as precipitants. The crystals belonged to space group P 2 1 2 1 2 and were most likely to contain six subunits in the asymmetric unit. The refined unit‐cell parameters were a = 164.46, b = 169.34, c = 60.79 Å. The crystals diffracted X‐rays to 2.1 Å resolution on a synchrotron‐radiation source and a three‐wavelength MAD data set has been collected. Determination of the chlorite dismutase structure will provide insights into the active site of the enzyme, for which no structures are currently available.