Crystallization and preliminary X-ray analysis of the small component of 4-hydroxyphenylacetate 3-monooxygenase (HpaC) and its cofactor complex from Thermus thermophilus HB8.

The small component of 4-hydroxyphenylacelate 3-monoxygenase (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 cluted HpaC protein solutions were separated into colourless and yellow-coloured fractions (i.e. apo-HpaC and HpaC-flavin complex, respectively). Crystals of apoHpaC 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 Å. 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 Å. Both crystals belong to the monoclinic system, space group P2[SUB1], 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 Å. They also belong to the monoclinic space group P2[SUB1]. 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. [ABSTRACT FROM AUTHOR]