Formate as the Main Branch Point for Methylotrophic Metabolism in Methylobacterium extorquens AM1

Abstract

ABSTRACT In serine cycle methylotrophs, methylene tetrahydrofolate (H 4 F) 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 H 4 F from formaldehyde during methylotrophic growth: one involving the reaction of formaldehyde with H 4 F to generate methylene H 4 F and the other involving conversion of formaldehyde to formate via methylene tetrahydromethanopterin-dependent enzymes and conversion of formate to methylene H 4 F via H 4 F-dependent enzymes. Evidence has suggested that the direct condensation reaction is the main source of methylene H 4 F during methylotrophic metabolism. However, mutants lacking enzymes that interconvert methylene H 4 F and formate are unable to grow on methanol, suggesting that this route for methylene H 4 F 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 H 4 F synthesis pathway through formate. These results suggested that the methylene H 4 F 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.