Isolation and purification of Thermus thermophilus HpaB by a crystallization approach

The oxygenase HpaB is a component of the 4-hydroxyphenylacetate 3-monooxygenase enzyme that is responsible for the hydroxylation of 4-hydroxy-phenylacetate. 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 I222, with unit-cell parameters a = 91.3, b = 99.8, c = 131.7 Å.