Abstract
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.
Highlights
A direct link between the Ln elements and microbial metabolism has been firmly established with the discovery of pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases (ADHs), from methylotrophic bacteria, that contain a Ln atom in the active site[1,2,3,4,5]
We further show that wild-type cell-free extracts from methanol plus La3+ grown cultures do not exhibit formaldehyde oxidation when measured by DCPIP reduction, and that the H4MPT pathway is required during Ln methylotrophy, providing in vivo evidence that XoxF1 methanol dehydrogenases (MDHs) does not oxidize methanol to formate in the periplasm
The protein was enriched by immobilized metal affinity chromatography (IMAC) and purified to homogeneity after histidine tag cleavage using recombinant tobacco etch virus protease[30,31] (Supplementary Fig. S1)
Summary
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[1,2,3,4,5]. Www.nature.com/scientificreports dephosphotetrahydromethanopterin (H4MPT) pathway and catalytic properties observed for XoxF MDH from Methylacidiphilum fumariolicum SolV revealed the capability to produce formate as the end product of periplasmic methanol oxidation[3]. In vivo evidence is suggestive of XoxF1-catalyzed formaldehyde oxidation in starving cells fed methanol[26] Since these studies were done in the absence of Ln, and due to the relevance of this enzyme for Ln-dependent methylotrophy and the scarcity of fundamental information available for Ln-dependent enzymes, with other Ln cofactors, a detailed kinetic study of XoxF1 MDH with Ln is needed. ExaF exhibits relatively low methanol dehydrogenase activity, but has the highest catalytic efficiency with ethanol of any reported PQQ-dependent EtDH. In vitro oxidation of methanol to formate by ExaF has been reported[5] This catalytic activity has not yet been corroborated in vivo
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