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-dichlorophenolindophenol (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. A direct link between the Ln elements and microbial metabolism has been firmly established with the discovery of PQQ-dependent alcohol dehydrogenases (ADHs), from methylotrophic bacteria, that contain a Ln atom in the active site 1-5. Thus far, Ln-PQQ ADHs can be grouped by their phylogeny and primary substrate as either XoxF-MDHs or ExaF-type ethanol dehydrogenases (EtDHs). MxaFI MDH has been considered the canonical primary catalyst for methanol oxidation in Gram-negative methylotrophs 6,7. The heterotetramer MxaFI contains PQQ that coordinates the calcium (Ca) ion 8-10. The discovery that Ln is incorporated into the active site of XoxF MDH in place of Ca, allowing catalytic function, has prompted the reexamination of methanol oxidation in methylotrophic bacteria 3,5,11-18. To date, only a few XoxF MDHs have been kinetically characterized 1,3,4,19,20. Phylogenetic analyses show there are at least five distinct families of XoxF MDHs 11,21 , and while it has been suggested that all XoxF MDHs may exhibit similar kinetic properties, reported data for these enzymes are currently inadequate to support such a broad characterization. In fact, recent studies have begun to identify differences in kinetic properties, cofactor usage, and pH optima of phylogenetically distinct XoxF enzymes 20,22,23. Lack of genes encoding the
