Crystallization and preliminary X-ray analysis of the small component of 4-hydroxyphenyl acetate 3-monooxygenase (HpaC) and its cofactor complex from Thermus thermophilus HR8

The small component of 4-hydroxyphenylacetate 3-monooxygenase (HpaC) is an NADH oxidoreductase containing a flavin molecule as a cofactor. HpaC reduces a flavin molecule and reduced flavin is subsequently supplied to the large component of 4-hydroxyphenylacetate 3-monooxygenase (HpaB). The HpaC protein from Thermus thermophilus HB8 has been overexpressed in Escherichia coli and crystallized. During purification, the eluted HpaC protein solutions were separated into colourless and yellow-coloured fractions (i.e. apo-HpaC and HpaC-flavin complex, respectively). Crystals of apo-HpaC grown in 5% (v/v) isopropyl alcohol, 0.1 M HEPES-NaOH pH 7.0, 40% (w/v) polyethylene glycol (PEG) 4000 and 10% (v/v) glycerol diffracted X-rays to a resolution of 1.85 Angstrom, whereas crystals of the HpaC-flavin complex grown in 20% (w/v) PEG 1000, 10% (w/v) PEG 8000 and 10% (v/v) glycerol diffracted X-rays to a resolution of 1.3 Angstrom. Both crystals belong to the monoclinic system, space group P2(1), with similar unit-cell parameters. Selenomethionyl protein crystals of the HpaC-flavin complex grown under similar conditions to the native crystals diffracted X-rays to a resolution of 1.8 Angstrom. They also belong to the monoclinic space group P2(1), but are not isomorphous to crystals of the HpaC-flavin complex of the native protein. MAD data for structure determination were successfully collected using these crystals.