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Title: Crystallization and preliminary X-ray analysis of the small component of 4-hydroxyphenylacetate 3-monooxygenase (HpaC) and its cofactor complex from Thermus thermophilus HB8.
Author: Kim SH, Miyatake H, Hisano T, Ohtani N, Miki K.
Journal: Acta Crystallogr D Biol Crystallogr; 2003 Dec; 59(Pt 12):2275-8. PubMed ID: 14646093.
Abstract:
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 A, 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 A. 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 A. 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.

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