Expression, crystallization and preliminary crystallographic analysis of human carbonyl reductase.
The cDNA of human placental carbonyl reductase (EC 1.1.1.184), a member of the short-chain dehydrogenase family of enzymes, was introduced into the plasmid vector pET-11a and the enzyme overexpressed in Escherichia coli. Recombinant carbonyl reductase was purified to homogeneity, characterized physically and kinetically, and crystallized for X-ray diffraction study. The recombinant protein was indistinguishable from human tissue carbonyl reductase (CR8.5 form) on the basis of partial sequence analysis, substrate specificity, susceptibility to inhibitors and immunochemical analysis. Similar to the tissue enzyme which which occurs in multiple molecular forms thought to arise from autocatalytic modification by 2-oxocarboxylic acids, a second form of the recombinant enzyme was generated under bacterial growth conditions producing high pyruvate concentrations. Purified recombinant protein, which corresponds to the smallest, most basic tissue form (CR8.5), was crystallized against 20% polyethyleneglycol 6000 in 25 mM 2-(N-morpholino)ethanesulfonic acid buffer (Mes) at pH 6.0 using the hanging drop method. Crystals of human carbonyl reductase diffract to better than 3.0 A, and the diffraction symmetry is consistent with a crystal that belongs to the tetragonal space group P4(1)(3)2(1)2 with unit cell dimensions of a = b = 55 A, c = 175 A, alpha = beta = gamma = 90.0. The asymmetric unit contains one molecule of 30.2 kDa.
Bohren
K M
KM
Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030.
Wermuth
B
B
Harrison
D
D
Ringe
D
D
Petsko
G A
GA
Gabbay
K H
KH
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D001483:Base Sequence; D003460:Crystallization; D018360:Crystallography, X-Ray; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D018076:DNA, Complementary; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase