Crystallization and preliminary X‐ray analysis of the oxygenase component (HpaB) of 4‐hydroxyphenylacetate 3‐monooxygenase from Thermus thermophilus HB8

The 4‐hydroxyphenylacetate (4HPA) 3‐monooxygenase enzyme catalyzes the hydroxylation of 4HPA to 3,4‐dihydroxyphenylacetate in the initial step of the degradation pathway of 4HPA. This enzyme consists of two components: an oxygenase (HpaB) and a reductase (HpaC). HpaB hydroxylates 4HPA using an oxygen molecule and a reduced flavin, which is supplied by HpaC. HpaB from Thermus thermophilus HB8 was overexpressed in Escherichia coli and crystallized. Crystals of HpaB were grown in 0.4  M 1,6‐hexanediol, 0.1  M sodium acetate pH 5.0 and 25%( v / v ) glycerol and diffracted X‐rays to a resolution of 1.60 Å. The crystals belong to the orthorhombic space group I 222, with unit‐cell parameters a = 91.8, b = 99.6, c = 131.1 Å. The asymmetric unit volume provides space for only one subunit of the tetrameric HpaB molecule, giving a Matthews coefficient V M of 2.8 Å 3  Da −1 and a solvent content of 55.1%. Platinum‐derivatized crystals of HpaB were prepared by soaking native crystals in a solution containing 1 m M ammonium tetrachloroplatinate(II) for 1 d and diffracted X‐rays to a resolution of 2.50 Å. MAD data were successfully collected for structural determination using these crystals.