Assessing Lanthanide-Dependent Methanol Dehydrogenase Activity: The Assay Matters

Artificial dye-coupled assays have been widely adopted as a rapid and convenient method to assess the activity of methanol dehydrogenases (MDH). Lanthanide(Ln)-dependent XoxF-MDHs from methanotrophs and methylotrophs are able to incorporate different lanthanides (Lns) in their active site. The artificial dye-coupled assay showed that the earlier Lns exhibit a higher enzyme activity than the late Lns. Although this assay is widely used, there are limitations. It is not unusual that a pH of 9 is required and that activators like ammonium have to be added to the assay mixture which do not reflect the conditions inside the cell. Moreover, different Ln-MDH variants are not obtained by the direct isolation from the cells grown with the respective Ln, but by metal titration of the Ln in a partial-apo-MDH or by incubation of an apo-MDH with the Ln. Herein, we report the cultivation of Ln-dependent methanotroph Methylacidiphilum fumariolicum SolV with nine different Lns, the isolation of the respective MDH and the assessment of the enzyme activity using the artificial dye-coupled assay. We compare these results with an adapted protein-coupled assay using the physiological partner and electron acceptor cytochrome c GJ (cyt c GJ ) instead of artificial dyes. We demonstrate that, depending on the assay, two distinct trends are observed among the Ln series. The specific activity of La-, Ce- and Pr-MDH, as measured by the protein-coupled assay, exceeds the specific enzyme activity measured by the dye-coupled assay. This suggests that the early Lns also have a positive effect on the interaction between XoxF-MDH and its cyt c GJ thereby increasing functional efficiency.