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 are able to incorporate different lanthanides (Lns) in their active site. Dye-coupled assays showed that the earlier Lns exhibit a higher enzyme activity than the late Lns. Despite widespread use, there are limitations: oftentimes a pH of 9 and activators are required for the assay. Moreover, Ln-MDH variants are not obtained by isolation from the cells grown with the respective Ln, but 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 MDHs and the assessment of the enzyme activity using the dye-coupled assay. We compare these results with a protein-coupled assay using its physiological electron acceptor cytochrome cGJ (cyt cGJ ). Depending on the assay, two distinct trends are observed among the Ln series. The specific enzyme activity of La-, Ce- and Pr-MDH, as measured by the protein-coupled assay, exceeds that measured by the dye-coupled assay. This suggests that early Lns also have a positive effect on the interaction between XoxF-MDH and its cyt cGJ thereby increasing functional efficiency.

Activity assays of methanol dehydrogenases.

The field of methanol dehydrogenases (MDHs) has experienced revival in the recent decade due to the observation of lanthanide-dependent MDH, in addition to widely known calcium-MDH. With the advent of lanthanide-dependent alcohol dehydrogenases, the need for reliable assays to evaluate and compare activities between different MDHs is obvious: from extremophilic to neutrophilic organisms, or with different lanthanide ions in the active site. Here we outline four assays that have been reported for Ln-MDH, discussing the advantages and disadvantages of the assays and their components. It should be noted, in 1990Day and Anthony produced a comprehensive summary in Methods in Enzymology on the available methods for Ca-MDH assays at the time (Day & Anthony, 1990). This chapter is an updated appraisal of the most important developments in the last 30years.