Investigation of lanthanide-dependent methylotrophy uncovers complementary roles for alcohol dehydrogenase enzymes

Abstract

ABSTRACTThe discovery that methylotrophic bacteria can utilize lanthanides as catalysts for methanol metabolism has opened new areas of biology and biochemistry. Recent studies of lanthanide-dependent enzymes have focused on biochemical and kinetic properties or the regulation of encoding genes. Kinetic analysis of a pyrroloquinoline quinone methanol dehydrogenase, XoxF1 (MexAM1_1746), from the model methylotrophMethylobacterium extorquensAM1 confirms the use of different lanthanides as cofactors and formaldehyde as a product of methanol oxidation, showing that not all XoxF MDH produce formate as the only end productin vivo. The dephosphotetrahydromethanopterin pathway for formaldehyde oxidation is still required for lanthanide-methylotrophic growth, as afaemutant does not grow with methanol in the presence of exogenous lanthanides. Increases of 15-22% in growth rate and 10-12.5% in growth yield are observed whenM. extorquensAM1 is grown in the presence of lanthanides with methanol. RNA-sequencing transcriptomics indicates remodeling of methanol, formaldehyde and formate oxidation gene expression, and targeted metabolomics shows increased accumulation of intracellular formate and decreased pools of several assimilatory intermediates. Methanol sensitivity growth assays show that the lanthanide-dependent pyrroloquinoline quinone alcohol dehydrogenase ExaF (MexAM1_1139), but not XoxF1, can reduce formaldehyde toxicity when lanthanides are present, providing evidence of a role for ExaF during lanthanide-dependent methylotrophy. We conclude from these results that lanthanide-dependent methylotrophy is more efficient than calcium-dependent methylotrophy inM. extorquensAM1, and that this change is due, at least in part, to the lanthanide-dependent enzymes XoxF1 and ExaF.IMPORTANCELanthanides serve as cofactors for pyrroloquinoline quinone containing alcohol dehydrogenase enzymes in methylotrophic bacteria. The present study addresses a fundamental gap in our understanding of how lanthanides impact metabolism, including a detailed assessment of the metabolic modifications to accommodate enhanced efficiency during methylotrophy. Kinetic characterization of XoxF1 provides a detailed description of the impact of diverse lanthanides on catalytic function for a lanthanide-dependent methanol dehydrogenase. We further show that the lanthanide-dependent ethanol dehydrogenase ExaF can oxidize formaldehydein vivo, revealing complementary roles for these enzymes. This study provides novel insight into the effects of lanthanides on bacterial metabolism, highlighting the implementation of multiple, redundant and complementary oxidation systems.