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 abstractLanthanide-dependent methanol dehydrogenases. Understanding the chemistry of artificial electron acceptors and redox dyes can yield more reproducible results.
Highlights
Biochemical assays are powerful analytical techniques used to identify or quantify proteins, to study the binding of substrates and inhibitors, and to measure the activity of enzymes
We stress here that the investigation of assay components phenazine ethosulfate (PES)/phenazine methosulfate (PMS) and DCPIP does not solve the problem with this so-called endogenous substrate of methanol dehydrogenase enzymes (MDH), but shall identify handling errors while performing colorimetric assays
This background reaction from endogenous substrate can be observed in PIPES buffer when MDH is assayed with its natural electron acceptor
Summary
Biochemical assays are powerful analytical techniques used to identify or quantify proteins, to study the binding of substrates and inhibitors, and to measure the activity of enzymes. The family of methanol dehydrogenase enzymes (MDH) has recently taken the spotlight again after it was discovered that many bacteria utilize lanthanide-dependent MDH of the XoxF family [1,2,3,4,5,6,7]. This finding has fueled an entirely new area of research—lanthanide-dependent bacterial metabolism and biochemistry. The electron transfer from the substrate, either methanol or formaldehyde, via the redox cofactor pyrroloquinoline quinone (PQQ) in the active site is coupled to electron acceptors (EA).
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