Expression, purification, crystallization and preliminary X-ray analysis of carbonyl reductase S1 from Candida magnoliae.
The NADPH-dependent carbonyl reductase S1 from Candida magnoliae stereoselectively catalyzes the reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate (CHBE), which is a chiral compound valuable as a building block for pharmaceuticals. Carbonyl reductase S1 was expressed in Escherichia coli and purified by Ni-affinity, ion-exchange and size-exclusion chromatography. Crystals of carbonyl reductase S1 were obtained by the sitting-drop vapour-diffusion method using PEG 400 as a precipitant. X-ray diffraction data were collected to 1.90 Å resolution using a synchrotron-radiation source. The crystals belonged to space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 77.7, c = 307.5 Å. The asymmetric unit contained two molecules of the protein, with a solvent content of 44.2%.
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Yoichi
Y
Suwa
Jun
J
Ohtsuka
Takuya
T
Miyakawa
Fabiana Lica
FL
Imai
Masahiko
M
Okai
Yoriko
Y
Sawano
Yoshihiko
Y
Yasohara
Michihiko
M
Kataoka
Sakayu
S
Shimizu
Masaru
M
Tanokura
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D002175:Candida / Q000201:enzymology*; D003460:Crystallization; D018360:Crystallography, X-Ray; D015870:Gene Expression
D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase