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Novel formaldehyde-activating enzyme in Methylobacterium extorquens AM1 required for growth on methanol.

ABSTRACT: Formaldehyde is toxic for all organisms from bacteria to humans due to its reactivity with biological macromolecules. Organisms that grow aerobically on single-carbon compounds such as methanol and methane face a special challenge in this regard because formaldehyde is a central metabolic intermediate during methylotrophic growth. In the alpha-proteobacterium Methylobacterium extorquens AM1, we found a previously unknown enzyme that efficiently catalyzes the removal of formaldehyde: it catalyzes the condensation of formaldehyde and tetrahydromethanopterin to methylene tetrahydromethanopterin, a reaction which also proceeds spontaneously, but at a lower rate than that of the enzyme-catalyzed reaction. Formaldehyde-activating enzyme (Fae) was purified from M. extorquens AM1 and found to be one of the major proteins in the cytoplasm. The encoding gene is located within a cluster of genes for enzymes involved in the further oxidation of methylene tetrahydromethanopterin to CO(2). Mutants of M. extorquens AM1 defective in Fae were able to grow on succinate but not on methanol and were much more sensitive toward methanol and formaldehyde. Uncharacterized orthologs to this enzyme are predicted to be encoded by uncharacterized genes from archaea, indicating that this type of enzyme occurs outside the methylotrophic bacteria.

SUBMITTER: Vorholt JA

PROVIDER: S-EPMC111405 | biostudies-literature | 2000 Dec

REPOSITORIES: biostudies-literature

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Novel formaldehyde-activating enzyme in Methylobacterium extorquens AM1 required for growth on methanol.

Vorholt J A JA Marx C J CJ Lidstrom M E ME Thauer R K RK

Journal of bacteriology 20001201 23

Formaldehyde is toxic for all organisms from bacteria to humans due to its reactivity with biological macromolecules. Organisms that grow aerobically on single-carbon compounds such as methanol and methane face a special challenge in this regard because formaldehyde is a central metabolic intermediate during methylotrophic growth. In the alpha-proteobacterium Methylobacterium extorquens AM1, we found a previously unknown enzyme that efficiently catalyzes the removal of formaldehyde: it catalyzes ...[more]

PMID: 11073907

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Project description:In order to provide information about the gene expression response that occurs when cells experience a change in carbon source, succinate limited chemostat cultures of Methylobacterium extorquens AM1 were grown to and maintained at an OD of ~0.63, transferred to flasks and methanol was added. Cells were harvested for RNA extraction at time: 0 min, 10 min, 30 min, 1 hr, 2 hr, 4 hr and 6 hr post transition. At 30 min, a no methanol addition sample was extracted as a carbon starvation control. These data were used in conjunction with flux, enzymatic and metabolite measurements to assess the changes in central metabolism during this transition. Abstract from manuscript: When organisms experience environmental change, how does their metabolic network reset and adapt to the new condition? This study focused on the mechanisms of metabolic adaptation occurring during the transition from succinate to methanol growth by the methylotrophic bacterium Methylobacterium extorquens, analyzing changes in carbon flux, gene expression, metabolites and enzymatic activities over time. Initially, cells experienced metabolic imbalance with excretion of metabolites, changes in nucleotide levels and cessation of cell growth. Though assimilatory pathways were induced rapidly, a transient block in carbon flow to biomass synthesis occurred, and enzymatic assays suggested methylenetetrahydrofolate dehydrogenase as one control point. This “downstream priming” mechanism ensures that significant carbon flux through these pathways does not occur until they are fully induced, precluding the buildup of toxic intermediates. Most metabolites that are required for growth on both carbon sources did not change significantly, even though transcripts and enzymatic activities required for their production changed radically, underscoring the concept of metabolic setpoints. Gene expression in succinate limited chemostat cultures was compared to gene expression in cells transferred to flasks before and after methanol addition. As a control, a time = 0 sample (RNA prepared from cells harvested directly from the chemostat) was compared to a time = 0 sample immediately obtained after the cells were transferred to flasks, before methanol was added in order to identify changes due to flask transfer. A carbon starvation control was also done comparing expression from time = 0 (chemostat cells) to cells transferred to flasks for 30 min with no carbon source added. Two biological replicates each with two techinal replicates (dye swap) were analyzed for time = 0 (chemostat) vs 10 min, 30 min, 1 hr and 2 hr after methanol addition. One biological replicate with two technical replicates (dye swap) were analyzed for time = 0 (chemostat) vs time = 0 (flask transfer), and time = 0 (chemostat) vs time = 4 hr, 6 hr and 30 min no methanol addition.

Dataset Information

Novel formaldehyde-activating enzyme in Methylobacterium extorquens AM1 required for growth on methanol.

Publications

Novel formaldehyde-activating enzyme in Methylobacterium extorquens AM1 required for growth on methanol.

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