Tanaka N, Nonaka T, Tanabe T, Yoshimoto T, Tsuru D, Mitsui Y
Department of BioEngineering, Nagaoka University of Technology, Niigata, Japan.
Biochemistry. 1996 Jun 18;35(24):7715-30. doi: 10.1021/bi951904d.
7 alpha-Hydroxysteroid dehydrogenase (7 alpha-HSDH;1 EC 1.1.1.159) is an NAD+-dependent oxidoreductase belonging to the short-chain dehydrogenase/reductase (SDR) 1 family. It catalyzes the dehydrogenation of a hydroxyl group at position 7 of the steroid skeleton of bile acids. The crystal structure of the binary (complexed with NAD+) complex of 7 alpha-HSDH has been solved at 2.3 A resolution by the multiple isomorphous replacement method. The structure of the ternary complex [the enzyme complexed with NADH, 7-oxoglycochenodeoxycholic acid (as a reaction product), and possibly partially glycochenodeoxycholic acid (as a substrate)] has been determined by a difference Fourier method at 1.8 A resolution. The enzyme 7 alpha-HSDH is an alpha/beta doubly wound protein having a Rossmann-fold domain for NAD (H) binding. Upon substrate binding, large conformation changes occur at the substrate binding loop (between the beta F strand and alpha G helix) and the C-terminal segment (residues 250-255). The variable amino acid sequences of the substrate-binding loop appear to be responsible for the wide variety of substrate specificities observed among the enzymes of the SDR family. The crystal structure of the ternary complex of 7 alpha-HSDH, which is the only structure available as the ternary complex among the enzymes of the SDR family, indicates that the highly conserved Tyr159 and Ser146 residues most probably directly interact with the hydroxyl group of the substrates although this observation cannot be definite due to an insufficiently characterized nature of the ternary complex. The strictly conserved Lys163 is hydrogen-bonded to both the 2'- and 3'-hydroxyl groups of the nicotinamide ribose of NAD(H). We propose a new catalytic mechanism possibly common to all the enzymes belonging to the SDR family in which a tyrosine residue (Tyr159) acts as a catalytic base and a serine residue (Ser146) plays a subsidiary role of stabilizing substrate binding.
7α-羟基类固醇脱氢酶（7α-HSDH；1 酶编号EC 1.1.1.159）是一种依赖烟酰胺腺嘌呤二核苷酸（NAD⁺）的氧化还原酶，属于短链脱氢酶/还原酶（SDR）1家族。它催化胆汁酸类固醇骨架7位上羟基的脱氢反应。7α-HSDH的二元复合物（与NAD⁺结合）的晶体结构已通过多重同晶置换法在2.3 Å分辨率下解析出来。三元复合物（该酶与NADH、7-氧代甘氨鹅去氧胆酸（作为反应产物）以及可能部分的甘氨鹅去氧胆酸（作为底物）结合）的结构已通过差值傅里叶法在1.8 Å分辨率下确定。酶7α-HSDH是一种α/β双绕蛋白，具有用于结合NAD（H）的罗斯曼折叠结构域。底物结合时，底物结合环（在βF链和αG螺旋之间）和C末端片段（第250 - 255位残基）会发生较大的构象变化。底物结合环可变的氨基酸序列似乎是造成SDR家族各酶之间观察到的多种底物特异性的原因。7α-HSDH三元复合物的晶体结构是SDR家族各酶中唯一可获得的三元复合物结构，表明高度保守的Tyr159和Ser146残基很可能直接与底物的羟基相互作用，尽管由于三元复合物的性质表征不足，这一观察结果并不确定。严格保守的Lys163与NAD（H）烟酰胺核糖的2'-和3'-羟基均形成氢键。我们提出一种可能为所有属于SDR家族的酶所共有的新催化机制，其中酪氨酸残基（Tyr159）作为催化碱，丝氨酸残基（Ser146）起到稳定底物结合的辅助作用。
