Department of Chemistry, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377, Munich, Germany.
Department of Microbiology, Institute of Wetland and Water Research, Radboud University, Nijmegen, The Netherlands.
J Biol Inorg Chem. 2020 Mar;25(2):199-212. doi: 10.1007/s00775-020-01752-9. Epub 2020 Feb 14.
Methanol dehydrogenases (MDH) have recently taken the spotlight with the discovery that a large portion of these enzymes in nature utilize lanthanides in their active sites. The kinetic parameters of these enzymes are determined with a spectrophotometric assay first described by Anthony and Zatman 55 years ago. This artificial assay uses alkylated phenazines, such as phenazine ethosulfate (PES) or phenazine methosulfate (PMS), as primary electron acceptors (EAs) and the electron transfer is further coupled to a dye. However, many groups have reported problems concerning the bleaching of the assay mixture in the absence of MDH and the reproducibility of those assays. Hence, the comparison of kinetic data among MDH enzymes of different species is often cumbersome. Using mass spectrometry, UV-Vis and electron paramagnetic resonance (EPR) spectroscopy, we show that the side reactions of the assay mixture are mainly due to the degradation of assay components. Light-induced demethylation (yielding formaldehyde and phenazine in the case of PMS) or oxidation of PES or PMS as well as a reaction with assay components (ammonia, cyanide) can occur. We suggest here a protocol to avoid these side reactions. Further, we describe a modified synthesis protocol for obtaining the alternative electron acceptor, Wurster's blue (WB), which serves both as EA and dye. The investigation of two lanthanide-dependent methanol dehydrogenases from Methylorubrum extorquens AM1 and Methylacidiphilum fumariolicum SolV with WB, along with handling recommendations, is presented. Lanthanide-dependent methanol dehydrogenases. Understanding the chemistry of artificial electron acceptors and redox dyes can yield more reproducible results.
甲醇脱氢酶（MDH）最近成为关注焦点，因为人们发现自然界中很大一部分这类酶在其活性部位使用镧系元素。这些酶的动力学参数是通过分光光度法测定的，该方法最早由 Anthony 和 Zatman 于 55 年前首次描述。这种人工测定方法使用烷基化吩嗪，如吩嗪乙硫酸盐（PES）或吩嗪甲硫酸盐（PMS）作为主要电子受体（EA），并且电子转移进一步与染料偶联。然而，许多研究小组报告了在没有 MDH 的情况下测定混合物褪色以及这些测定重现性的问题。因此，不同物种的 MDH 酶之间的动力学数据的比较通常很繁琐。使用质谱、紫外可见分光光度法和电子顺磁共振（EPR）光谱法，我们表明测定混合物的副反应主要是由于测定成分的降解。光诱导去甲基化（在 PMS 的情况下生成甲醛和吩嗪）或 PES 或 PMS 的氧化以及与测定成分（氨、氰化物）的反应都可能发生。我们在这里提出了一种避免这些副反应的方案。此外，我们描述了一种改良的替代电子受体，Wurster's blue（WB）的合成方案，它既可以作为 EA，也可以作为染料。我们描述了使用 WB 对两种来自 Methylorubrum extorquens AM1 和 Methylacidiphilum fumariolicum SolV 的镧系依赖性甲醇脱氢酶的研究，以及处理建议。镧系依赖性甲醇脱氢酶。了解人工电子受体和氧化还原染料的化学性质可以产生更可重现的结果。
