Rozeboom Henriëtte J, Yu Shukun, Mikkelsen Rene, Nikolaev Igor, Mulder Harm J, Dijkstra Bauke W
Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands.
DuPont Industrial Biosciences, Brabrand, Aarhus, Denmark.
Protein Sci. 2015 Dec;24(12):2044-54. doi: 10.1002/pro.2818. Epub 2015 Oct 20.
The quinone-dependent alcohol dehydrogenase (PQQ-ADH, E.C. 1.1.5.2) from the Gram-negative bacterium Pseudogluconobacter saccharoketogenes IFO 14464 oxidizes primary alcohols (e.g. ethanol, butanol), secondary alcohols (monosaccharides), as well as aldehydes, polysaccharides, and cyclodextrins. The recombinant protein, expressed in Pichia pastoris, was crystallized, and three-dimensional (3D) structures of the native form, with PQQ and a Ca(2+) ion, and of the enzyme in complex with a Zn(2+) ion and a bound substrate mimic were determined at 1.72 Å and 1.84 Å resolution, respectively. PQQ-ADH displays an eight-bladed β-propeller fold, characteristic of Type I quinone-dependent methanol dehydrogenases. However, three of the four ligands of the Ca(2+) ion differ from those of related dehydrogenases and they come from different parts of the polypeptide chain. These differences result in a more open, easily accessible active site, which explains why PQQ-ADH can oxidize a broad range of substrates. The bound substrate mimic suggests Asp333 as the catalytic base. Remarkably, no vicinal disulfide bridge is present near the PQQ, which in other PQQ-dependent alcohol dehydrogenases has been proposed to be necessary for electron transfer. Instead an associated cytochrome c can approach the PQQ for direct electron transfer.
革兰氏阴性菌产糖假葡萄糖杆菌IFO 14464中的醌依赖性醇脱氢酶（PQQ-ADH，E.C. 1.1.5.2）可氧化伯醇（如乙醇、丁醇）、仲醇（单糖）以及醛、多糖和环糊精。在毕赤酵母中表达的重组蛋白被结晶，并分别在1.72 Å和1.84 Å分辨率下确定了天然形式（含有PQQ和Ca(2+)离子）以及与Zn(2+)离子和结合底物类似物形成复合物的酶的三维（3D）结构。PQQ-ADH呈现出八叶β-螺旋桨折叠结构，这是I型醌依赖性甲醇脱氢酶的特征。然而，Ca(2+)离子的四个配体中有三个与相关脱氢酶的配体不同，且它们来自多肽链的不同部分。这些差异导致活性位点更加开放、易于接近，这就解释了为什么PQQ-ADH能够氧化多种底物。结合的底物类似物表明Asp333是催化碱基。值得注意的是，在PQQ附近不存在邻位二硫键桥，而在其他PQQ依赖性醇脱氢酶中，该二硫键桥被认为是电子转移所必需的。相反，一个相关的细胞色素c可以靠近PQQ进行直接电子转移。
