Höffken H Wolfgang, Duong Minh, Friedrich Thomas, Breuer Michael, Hauer Bernhard, Reinhardt Richard, Rabus Ralf, Heider Johann
BASF AG, Physical Chemistry and Informatics, 67056 Ludwigshafen, Germany.
Biochemistry. 2006 Jan 10;45(1):82-93. doi: 10.1021/bi051596b.
(S)-1-Phenylethanol dehydrogenase (PED) from the denitrifying bacterium strain EbN1 catalyzes the NAD+-dependent, stereospecific oxidation of (S)-1-phenylethanol to acetophenone and the biotechnologically interesting reverse reaction. This novel enzyme belongs to the short-chain alcohol dehydrogenase/aldehyde reductase family. The coding gene (ped) was heterologously expressed in Escherichia coli and the purified protein was crystallized. The X-ray structures of the apo-form and the NAD+-bound form were solved at a resolution of 2.1 and 2.4 A, respectively, revealing that the enzyme is a tetramer with two types of hydrophobic dimerization interfaces, similar to beta-oxoacyl-[acyl carrier protein] reductase (FabG) from E. coli. NAD+-binding is associated with a conformational shift of the substrate binding loop of PED from a crystallographically unordered "open" to a more ordered "closed" form. Modeling the substrate acetophenone into the active site revealed the structural prerequisites for the strong enantioselectivity of the enzyme and for the catalytic mechanism. Studies on the steady-state kinetics of PED indicated a highly positive cooperativity of both catalytic directions with respect to the substrates. This is contrasted by the behavior of FabG. Moreover, PED exhibits extensive regulation on the enzyme level, being inhibited by elevated concentrations of substrates and products, as well as the wrong enantiomer of 1-phenylethanol. These regulatory properties of PED are consistent with the presence of a putative "transmission module" between the subunits. This module consists of the C-terminal loops of all four subunits, which form a special interconnected structural domain and mediate close contact of the subunits, and of a phenylalanine residue in each subunit that reaches out between substrate-binding loop and C-terminal domain of an adjacent subunit. These elements may transmit the substrate-induced conformational change of the substrate binding loop from one subunit to the others in the tetrameric complex and thus mediate the cooperative behavior of PED.
反硝化细菌菌株EbN1中的（S）-1-苯乙醇脱氢酶（PED）催化（S）-1-苯乙醇依赖NAD +的立体特异性氧化生成苯乙酮以及具有生物技术意义的逆反应。这种新型酶属于短链醇脱氢酶/醛还原酶家族。编码基因（ped）在大肠杆菌中进行了异源表达，并对纯化后的蛋白进行了结晶。分别以2.1 Å和2.4 Å的分辨率解析了无辅基形式和NAD +结合形式的X射线结构，结果表明该酶是一种四聚体，具有两种类型的疏水二聚化界面，类似于大肠杆菌中的β-氧代酰基-[酰基载体蛋白]还原酶（FabG）。NAD +的结合与PED底物结合环的构象变化有关，从晶体学上无序的“开放”形式转变为更有序的“闭合”形式。将底物苯乙酮模拟到活性位点揭示了该酶强烈对映选择性和催化机制的结构前提。对PED稳态动力学的研究表明，两个催化方向对底物均具有高度正协同性。这与FabG的行为形成对比。此外，PED在酶水平上表现出广泛的调节作用，受到底物和产物浓度升高以及1-苯乙醇错误对映体的抑制。PED的这些调节特性与亚基之间存在假定的“传递模块”一致。该模块由所有四个亚基的C末端环组成，这些环形成一个特殊的相互连接的结构域并介导亚基之间的紧密接触，还包括每个亚基中的一个苯丙氨酸残基，该残基在底物结合环和相邻亚基的C末端结构域之间伸出。这些元件可能将底物诱导的底物结合环构象变化从四聚体复合物中的一个亚基传递到其他亚基，从而介导PED的协同行为。
