Assessing Lanthanide-Dependent Methanol Dehydrogenase Activity: The Assay Matters

Abstract

AbstractArtificial 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 methanotrophMethylacidiphilum fumariolicumSolV 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 cytochromecGJ(cytcGJ) 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 cytcGJthereby increasing functional efficiency.