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-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.
Kim
Seong-Hoon
SH
RIKEN Harima Institute/SPring-8, Koto 1-1-1, Mikazuki-cho, Sayo-gun, Hyogo 679-5148, Japan.
Miyatake
Hideyuki
H
Hisano
Tamao
T
Ohtani
Naoto
N
Miki
Kunio
K
D001059:Apoproteins / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003460:Crystallization / Q000379:methods; D018360:Crystallography, X-Ray / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology; D005415:Flavins / Q000737:chemistry* / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012645:Selenomethionine / Q000737:chemistry; D016963:Thermus thermophilus / Q000201:enzymology*
D001059:Apoproteins; D001426:Bacterial Proteins; D005415:Flavins; D011994:Recombinant Proteins; D012645:Selenomethionine; D006899:Mixed Function Oxygenases; C026252:4-hydroxyphenylacetate 3-monooxygenase