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Lanthanides are utilized by microbial methanol dehydrogenases, and it has been proposed that lanthanides may be important for other type I alcohol dehydrogenases. A triple mutant strain ( mxaF xoxF1 xoxF2 ; named MDH-3), deficient in the three known methanol dehydrogenases of the model methylotroph Methylobacterium extorquens AM1, is able to grow poorly with methanol if exogenous lanthanides are added to the growth medium. When the gene encoding a putative quinoprotein ethanol dehydrogenase, exaF , was mutated in the MDH-3 background, the quadruple mutant strain could no longer grow on methanol in minimal medium with added lanthanum (La 3+ ). ExaF was purified from cells grown with both calcium (Ca 2+ ) and La 3+ and with Ca 2+ only, and the protein species were studied biochemically. Purified ExaF is a 126-kDa homodimer that preferentially binds La 3+ over Ca 2+ in the active site. UV-visible spectroscopy indicates the presence of pyrroloquinoline quinone (PQQ) as a cofactor. ExaF purified from the Ca 2+ -plus-La 3+ condition readily oxidizes ethanol and has secondary activities with formaldehyde, acetaldehyde, and methanol, whereas ExaF purified from the Ca 2+ -only condition has minimal activity with ethanol as the substrate and activity with methanol is not detectable. The exaF mutant is not affected for growth with ethanol; however, kinetic and in vivo data show that ExaF contributes to ethanol metabolism when La 3+ is present, expanding the role of lanthanides to multicarbon metabolism. IMPORTANCE ExaF is the most efficient PQQ-dependent ethanol dehydrogenase reported to date and, to our knowledge, the first non-XoxF-type alcohol oxidation system reported to use lanthanides as a cofactor, expanding the importance of lanthanides in biochemistry and bacterial metabolism beyond methanol dehydrogenases to multicarbon metabolism. These results support an earlier proposal that an aspartate residue near the catalytic aspartate residue may be an indicator of rare-earth element utilization by type I alcohol dehydrogenases.

Lanthanide (Ln) elements are utilized as cofactors for catalysis by XoxF-type methanol dehydrogenases (MDHs). A primary assumption is that XoxF enzymes produce formate from methanol oxidation, which could impact organisms that require formaldehyde for assimilation. We report genetic and phenotypic evidence showing that XoxF1 (MexAM1_1740) from Methylobacterium extorquens AM1 produces formaldehyde, and not formate, during growth with methanol. Enzyme purified with lanthanum or neodymium oxidizes formaldehyde. However, formaldehyde oxidation via 2,6-dichlorophenol-indophenol (DCPIP) reduction is not detected in cell-free extracts from wild-type strain methanol- and lanthanum-grown cultures. Formaldehyde activating enzyme (Fae) is required for Ln methylotrophic growth, demonstrating that XoxF1-mediated production of formaldehyde is essential. Addition of exogenous lanthanum increases growth rate with methanol by 9–12% but does not correlate with changes to methanol consumption or formaldehyde accumulation. Transcriptomics analysis of lanthanum methanol growth shows upregulation of xox1 and downregulation of mxa genes, consistent with the Ln-switch, no differential expression of formaldehyde conversion genes, downregulation of pyrroloquinoline quinone (PQQ) biosynthesis genes, and upregulation of fdh4 formate dehydrogenase (FDH) genes. Additionally, the Ln-dependent ethanol dehydrogenase ExaF reduces methanol sensitivity in the fae mutant strain when lanthanides are present, providing evidence for the capacity of an auxiliary role for ExaF during Ln-dependent methylotrophy.

The 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.