Wang Shanshan, Nie Yao, Yan Xu, Ko Tzu-Ping, Huang Chun-Hsiang, Chan Hsiu-Chien, Guo Rey-Ting, Xiao Rong
School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, People's Republic of China.
Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.
Acta Crystallogr F Struct Biol Commun. 2014 Jun;70(Pt 6):800-2. doi: 10.1107/S2053230X1400908X. Epub 2014 May 24.
The NADH-dependent (R)-carbonyl reductase from Candida parapsilosis (RCR) catalyzes the asymmetric reduction of 2-hydroxyacetophenone (HAP) to produce (R)-1-phenyl-1,2-ethanediol [(R)-PED], which is used as a versatile building block for the synthesis of pharmaceuticals and fine chemicals. To gain insight into the catalytic mechanism, the structures of complexes of RCR with ligands, including the coenzyme, are important. Here, the recombinant RCR protein was expressed and purified in Escherichia coli and was crystallized in the presence of NAD+. The crystals, which belonged to the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a=85.64, b=106.11, c=145.55 Å, were obtained by the sitting-drop vapour-diffusion method and diffracted to 2.15 Å resolution. Initial model building indicates that RCR forms a homotetramer, consistent with previous reports of medium-chain-type alcohol dehydrogenases.
近平滑假丝酵母依赖烟酰胺腺嘌呤二核苷酸（NADH）的（R）-羰基还原酶（RCR）催化2-羟基苯乙酮（HAP）的不对称还原反应，生成（R）-1-苯基-1,2-乙二醇[（R）-PED]，（R）-PED可用作合成药物和精细化学品的通用结构单元。为深入了解催化机制，RCR与包括辅酶在内的配体形成的复合物结构至关重要。在此，重组RCR蛋白在大肠杆菌中表达并纯化，且在NAD⁺存在的情况下结晶。通过坐滴气相扩散法获得了属于正交晶系空间群P2₁2₁2₁的晶体，其晶胞参数为a = 85.64、b = 106.11、c = 145.55 Å，衍射分辨率达到2.15 Å。初始模型构建表明，RCR形成同四聚体，这与先前关于中链型醇脱氢酶的报道一致。
