Bohren K M, Wermuth B, Harrison D, Ringe D, Petsko G A, Gabbay K H
Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030.
J Mol Biol. 1994 Dec 16;244(5):659-64. doi: 10.1006/jmbi.1994.1762.
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.
人胎盘羰基还原酶（EC 1.1.1.184）属于短链脱氢酶家族，其互补DNA（cDNA）被导入质粒载体pET - 11a，并在大肠杆菌中实现该酶的过表达。重组羰基还原酶被纯化至同质，进行了物理和动力学特性分析，并结晶用于X射线衍射研究。基于部分序列分析、底物特异性、对抑制剂的敏感性和免疫化学分析，该重组蛋白与人组织羰基还原酶（CR8.5形式）无法区分。与组织酶类似，组织酶以多种分子形式存在，被认为是由2 - 氧代羧酸的自催化修饰产生的，在产生高丙酮酸浓度的细菌生长条件下，重组酶也产生了第二种形式。使用悬滴法，将对应于最小、最基本组织形式（CR8.5）的纯化重组蛋白，在pH 6.0的25 mM 2 -（N - 吗啉代）乙磺酸缓冲液（Mes）中，与20%聚乙二醇6000一起结晶。人羰基还原酶晶体的衍射分辨率优于3.0 Å，衍射对称性与属于四方晶系空间群P4(1)(3)2(1)2的晶体一致，晶胞参数为a = b = 55 Å，c = 175 Å，α = β = γ = 90.0。不对称单元包含一个30.2 kDa的分子。
