Assessing Lanthanide-Dependent Methanol Dehydrogenase Activity: The Assay Matters

Area of Science:

• Biochemistry
• Microbiology
• Enzymology

Background:

• Methanol dehydrogenases (MDH) are crucial enzymes, often assessed using artificial dye-coupled assays.
• Lanthanide (Ln)-dependent XoxF-MDHs can incorporate different Lns, with dye assays suggesting early Lns yield higher activity.
• Current methods require specific pH and activators, and Ln-MDH variants are typically generated in vitro, not via direct cultivation.

Purpose of the Study:

• To investigate the cultivation of Ln-dependent methanotrophs with various Lns.
• To assess the activity of isolated Ln-MDHs using both dye-coupled and protein-coupled assays.
• To compare enzyme activity trends between the two assay types and understand Ln effects on enzyme-substrate interactions.

Main Methods:

• Cultivation of Methylacidiphilum fumariolicum SolV with nine different Lns.
• Isolation of the respective Ln-dependent XoxF-MDH variants.
• Enzyme activity assessment using traditional dye-coupled assays and a protein-coupled assay with cytochrome c_GJ (cyt c_GJ).

Main Results:

• Two distinct activity trends were observed depending on the assay method used.
• The protein-coupled assay showed higher specific enzyme activity for La-, Ce-, and Pr-MDH compared to the dye-coupled assay.
• Early Lns (La, Ce, Pr) appear to positively influence the interaction between XoxF-MDH and its physiological electron acceptor, cyt c_GJ.

Conclusions:

• Direct cultivation and isolation of Ln-MDHs provide insights into their activity.
• The choice of assay significantly impacts the observed trends in Ln-MDH activity.
• Early lanthanides enhance the functional efficiency of XoxF-MDH by promoting interaction with its native electron acceptor.