Formate as the main branch point for methylotrophic metabolism in Methylobacterium extorquens AM1

Abstract

In serine cycle methylotrophs, methylene tetrahydrofolate (H4F) is the entry point of reduced one-carbon compounds into the serine cycle for carbon assimilation during methylotrophic metabolism. In these bacteria, two routes are possible for generating methylene H4F from formaldehyde during methylotrophic growth: one involving the reaction of formaldehyde with H4F to generate methylene H4F and the other involving conversion of formaldehyde to formate via methylene tetrahydromethanopterin-dependent enzymes and conversion of formate to methylene H4F via H4F-dependent enzymes. Evidence has suggested that the direct condensation reaction is the main source of methylene H4F during methylotrophic metabolism. However, mutants lacking enzymes that interconvert methylene H4F and formate are unable to grow on methanol, suggesting that this route for methylene H4F synthesis should have a significant role in biomass production during methylotrophic metabolism. This problem was investigated in Methylobacterium extorquens AM1. Evidence was obtained suggesting that the existing deuterium assay might overestimate the flux through the direct condensation reaction. To test this possibility, it was shown that only minor assimilation into biomass occurred in mutants lacking the methylene H4F synthesis pathway through formate. These results suggested that the methylene H4F synthesis pathway through formate dominates assimilatory flux. A revised kinetic model was used to validate this possibility, showing that physiologically plausible parameters in this model can account for the metabolic fluxes observed in vivo. These results all support the suggestion that formate, not formaldehyde, is the main branch point for methylotrophic metabolism in M. extorquens AM1.

FIG. 1.

Traditional scheme of methylotrophic metabolism in serine cycle methylotrophs (12, 19). CH₂=H₄F, methylene tetrahydrofolate; CH₂=H₄MPT, methylene H₄MPT.

FIG. 2.

The assay for methylene-tetrahydrofolate synthesis routes in whole cells using deuterated methanol (CD₃OD). (A) Spontaneous condensation consists of joining formaldehyde (in this case, DCDO) and H₄F to form doubly deuterated methylene-tetrahydrofolate (CD₂=H₄F). In the alternate pathway, the one-carbon unit is joined to H₄MPT, released as formate (DCOO⁻), and then joined to H₄F, leading to the formation of CDH=H₄F. In this scheme, no significant NADPH (NADPD in this experiment) is produced from the H₄MPT pathway, and the ratio of doubly deuterated (D₂) serine to singly deuterated (D) serine reflects relative fluxes through the spontaneous and alternate pathways. (B) In this scheme, it is assumed that significant NADPH (NADPD in this experiment) is generated by the H₄MPT pathway. Contamination of the NADPH pool with deuterated NADPH produced after metabolism of deuterated methanol will increase the amount of +2 serine at the expense of +1 serine, leading to an overestimation of flux through spontaneous condensation. (C) Effect of assay incubation time on the ratio of +2 serine to +1 serine in methanol-grown cells.

FIG. 3.

Distribution of fluxes (in mM/s) in wild-type M. extorquens AM1 chemostat cells grown on methanol, assuming that no methylene H₄F is produced via spontaneous condensation.

FIG. 4.

Proposed scheme of methylotrophic metabolism in M. extorquens AM1 showing formate (HCOO⁻) as the branch point between assimilatory and dissimilatory metabolism.