Crystallization and preliminary X-ray crystallographic studies of dissimilatory nitrite reductase isolated from Hyphomicrobium denitrificans A3151.
Dissimilatory nitrite reductase isolated from Hyphomicrobium denitrificans A3151 (HdNIR) is a novel copper-containing nitrite reductase (CuNIR) composed of six identical subunits. One plastocyanin-like domain and one green CuNIR-like domain are connected to each other, suggesting that the HdNIR subunit structure resembles a complex of green CuNIR and pseudoazurin (or azurin). Recombinant HdNIR protein was crystallized using the hanging-drop vapour-diffusion method with PEG 4000 as the precipitant at pH 8.9. X-ray diffraction data were collected to 2.35 A resolution. The HdNIR crystal belonged to the tetragonal space group P4(1) (or P4(3)), with unit-cell parameters a = b = 221.9, c = 165.2 A, giving 12 molecules (two hexamers) per asymmetric unit and a solvent content of 64%. A mutant form of HdNIR, C260A, which lacks the type I copper ion in the CuNIR-like domain, was prepared and crystallized under wild-type HdNIR conditions. The C260A mutant crystal belonged to the cubic space group P4(3)32 (or P4(1)32), with unit-cell parameters a = b = c = 153.7 A, giving one molecule per asymmetric unit and a solvent content of 59%. X-ray diffraction data were collected to 3.5 A resolution. To solve the crystal structure of HdNIR, the multiwavelength anomalous dispersion (MAD) method and the molecular-replacement method are currently being used.
Xie
Yong
Y
Department of Materials Chemistry, Graduate School of Engineering, Osaka University 1-2, Yamadaoka, Suita, Osaka 565-0871, Japan.
Inoue
Tsuyoshi
T
Seike
Nozomu
N
Matsumura
Hiroyoshi
H
Kanbayashi
Koji
K
Itoh
Koushi
K
Kataoka
Kunisige
K
Yamaguchi
Kazuya
K
Suzuki
Shinnichiro
S
Kai
Yasushi
Y
D003460:Crystallization; D018360:Crystallography, X-Ray; D003580:Cytochromes; D004058:Diffusion; D003576:Electron Transport Complex IV / Q000737:chemistry*; D020582:Hyphomicrobium / Q000201:enzymology*; D009154:Mutation; D009572:Nitrite Reductases / Q000737:chemistry*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
D003580:Cytochromes; D009572:Nitrite Reductases; C028123:nitrite reductase (NO-forming); D003576:Electron Transport Complex IV