EXPRESSION, CRYSTALLIZATION AND PRELIMINARY CRYSTALLOGRAPHIC ANALYSISOF HUMAN CARBONYL REDUCTASE

The cDNA of human placental carbonyl reductase (EC 1.1.1.184), a membe r of the short- chain dehydrogenase family of enzymes, was introduced into the plasmid vector pET-11a and the enzyme overexpressed in Escher ichia 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 h uman tissue carbonyl reductase (CR(8.5) form) on the basis of partial sequence analysis, substrate specificity, susceptibility to inhibitors and immunochemical analysis. Similar to the tissue enzyme which occur s in multiple molecular forms thought to arise from autocatalytic modi fication by 2-oxocarboxylic acids, a second form of the recombinant en zyme was generated under bacterial growth conditions producing high py ruvate concentrations. Purified recombinant protein, which corresponds to the smallest, most basic tissue form (CR(8.5)), was crystallized a gainst 20% polyethgrleneglycol 6000 in 25 mM 2-(N-morpholino)ethanesul fonic acid buffer (Mes) at pH 6.0 using the hanging drop method. Cryst als of human carbonyl reductase diffract to better than 3.0 Angstrom, 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 Angstrom, c = 175 Angstrom, alpha = beta = gamma = 90.0 . The asymmetric unit contains one molecule of 30.2 kDa.