Abstract

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.

Graphic abstract

Lanthanide-dependent methanol dehydrogenases. Understanding the chemistry of artificial electron acceptors and redox dyes can yield more reproducible results.

中文翻译：

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了解甲醇脱氢酶测定中人工电子受体 PES、PMS、DCPIP 和沃斯特蓝的化学性质。

摘要

甲醇脱氢酶（MDH）最近引起了人们的关注，因为我们发现自然界中大部分酶的活性位点都利用了镧系元素。这些酶的动力学参数是通过 Anthony 和 Zatman 55 年前首次描述的分光光度测定法测定的。这种人工测定使用烷基化吩嗪，例如吩嗪硫酸乙酯 (PES) 或吩嗪硫酸甲酯 (PMS)，作为主要电子受体 (EA)，电子转移进一步与染料偶联。然而，许多团体报告了有关在不存在 MDH 的情况下测定混合物漂白以及这些测定的再现性的问题。因此，不同物种的 MDH 酶之间的动力学数据比较通常很麻烦。使用质谱、紫外-可见光和电子顺磁共振（EPR）光谱，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。可能发生光诱导的去甲基化（在 PMS 的情况下产生甲醛和吩嗪）或 PES 或 PMS 的氧化以及与测定成分（氨、氰化物）的反应。我们在此建议一个避免这些副反应的方案。此外，我们描述了一种改进的合成方案，用于获得替代电子受体沃斯特蓝 (WB)，它既充当 EA 又充当染料。本文介绍了使用 WB 对来自Mmethylorubrum extorquens AM1 和Mmethylacidiphilum fumariolicum SolV的两种镧系元素依赖性甲醇脱氢酶进行的研究以及处理建议。

图文摘要

镧系元素依赖性甲醇脱氢酶。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具重复性的结果。