Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, 2238522 Yokohama, Japan.
Chemistry. 2011 Jan 10;17(2):557-63. doi: 10.1002/chem.201001924. Epub 2010 Nov 9.
Variant G74C of arylmalonate decarboxylase (AMDase) from Bordatella bronchoseptica has a unique racemising activity towards profens. By protein engineering, variant G74C/V43A with a 20-fold shift towards promiscuous racemisation was obtained, based on a reduced activity in the decarboxylation reaction and a two-fold increase in the racemisation activity. The mutant showed an extended substrate range, with a 30-fold increase in the reaction rate towards ketoprofen. Molecular dynamics simulations and the substrate profile of the racemase indicate that the steric and polar effects of the substrate structure play a more dominant role on catalysis than mere kinetic α-proton acidity. The observation that the conversion of β,γ-unsaturated carboxylic acids does not lead to a rearrangement to form their α,β isomers indicates a concerted rather than a stepwise mechanism. Interestingly, a substrate bearing a nitro group instead of the carboxylic acid group on the α-carbon atom was also converted by the racemase.
从支气管败血波氏杆菌中分离出的芳基丙二酸 decarboxylase（AMDase）的变体 G74C 对 profens 具有独特的外消旋化活性。通过蛋白质工程，基于在脱羧反应中的活性降低和外消旋化活性增加两倍，获得了变体 G74C/V43A，其对混杂的外消旋化具有 20 倍的转变。该突变体显示出扩展的底物范围，对酮洛芬的反应速率增加了 30 倍。分子动力学模拟和外消旋酶的底物谱表明，底物结构的空间和极性效应对催化的作用比单纯的动力学α-质子酸度更为重要。观察到β,γ-不饱和羧酸的转化不会导致重排形成其α,β异构体，表明是协同而不是逐步的机制。有趣的是，带有硝基取代羧酸基团的α-碳原子的底物也被外消旋酶转化。
