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Crystallization and preliminary X-ray analysis of the small component of 4-hydroxyphenylacetate 3-monooxygenase (HpaC) and its cofactor complex from Thermus thermophilus HB8.
- Source :
-
Acta Crystallographica: Section D (Wiley-Blackwell) . Dec2003, Vol. 59 Issue 12, p2275-2278. 4p. - Publication Year :
- 2003
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Abstract
- 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]
Details
- Language :
- English
- ISSN :
- 09074449
- Volume :
- 59
- Issue :
- 12
- Database :
- Academic Search Index
- Journal :
- Acta Crystallographica: Section D (Wiley-Blackwell)
- Publication Type :
- Academic Journal
- Accession number :
- 11795045
- Full Text :
- https://doi.org/10.1107/S0907444903020821