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Title
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Crystallization and preliminary X-ray analysis of the oxygenase component (HpaB) of 4-hydroxyphenylacetate 3-monooxygenase from Thermus thermophilus HB8.
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Authors
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S.H.Kim,
H.Miyatake,
T.Hisano,
W.Iwasaki,
A.Ebihara,
K.Miki.
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Ref.
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Acta Crystallogr Sect F Struct Biol Cryst Commun, 2007,
63,
556-559.
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PubMed id
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Abstract
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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 A. The
crystals belong to the orthorhombic space group I222, with unit-cell parameters
a = 91.8, b = 99.6, c = 131.1 A. The asymmetric unit volume provides space for
only one subunit of the tetrameric HpaB molecule, giving a Matthews coefficient
V(M) of 2.8 A3 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 mM ammonium tetrachloroplatinate(II) for 1 d and diffracted X-rays
to a resolution of 2.50 A. MAD data were successfully collected for structural
determination using these crystals.
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