Open AccessIsolation and purification of Thermus thermophilus HpaB by a crystallization approach
Author(s) -
Soulimane Tewfik,
O'Kane Sarah R.,
Kolaj Olga
Publication year - 2010
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309110003714
Subject(s) - thermus thermophilus , crystallization , chemistry , oxygenase , enzyme , thermus , stereochemistry , thermophile , crystallography , biochemistry , escherichia coli , organic chemistry , gene
The oxygenase HpaB is a component of the 4‐hydroxyphenylacetate 3‐monooxygenase enzyme that is responsible for the hydroxylation of 4‐hydroxyphenylacetate. It utilizes molecular oxygen and a reduced flavin, which is provided by HpaC, the second component of the enzyme. While isolating integral membrane respiratory complexes from Thermus thermophilus , microcrystals of HpaB were formed. Further purification of the enzyme was achieved by repetitive crystallization. Subsequently, well shaped single crystals of the native enzyme that diffract to 1.82 Å resolution were grown in sitting drops. They belong to the orthorhombic space group I 222, with unit‐cell parameters a = 91.3, b = 99.8, c = 131.7 Å.