Purification of the Formate-Tetrahydrofolate Ligasefrom Methylobacterium extorquens AM1 and Demonstrationof Its Requirement for MethylotrophicGrowth

Abstract

The serine cycle methylotroph Methylobacterium extorquens AM1 contains two pterin-dependent pathways for C 1 transfers, the tetrahydrofolate (H 4 F) pathway and the tetrahydromethanopterin (H 4 MPT) pathway, and both are required for growth on C 1 compounds. With the exception of formate-tetrahydrofolate ligase (FtfL, alternatively termed formyl-H 4 F synthetase), all of the genes encoding the enzymes comprising these two pathways have been identified, and the corresponding gene products have been purified and characterized. We present here the purification and characterization of FtfL from M. extorquens AM1 and the confirmation that this enzyme is encoded by an ftfL homolog identified previously through transposon mutagenesis. Phenotypic analyses of the ftfL mutant strain demonstrated that FtfL activity is required for growth on C 1 compounds. Unlike mutants defective for the H 4 MPT pathway, the ftfL mutant strain does not exhibit phenotypes indicative of defective formaldehyde oxidation. Furthermore, the ftfL mutant strain remained competent for wild-type conversion of [ 14 C]methanol to [ 14 C]CO 2 . Collectively, these data confirm our previous presumptions that the H 4 F pathway is not the key formaldehyde oxidation pathway in M. extorquens AM1. Rather, our data suggest an alternative model for the role of the H 4 F pathway in this organism in which it functions to convert formate to methylene H 4 F for assimilatory metabolism.

Genomic Analysis of Carbon Source Metabolism of Shewanella oneidensis MR-1: Predictions versus Experiments

“…Enzyme complexes are indicated by SO numbers separated by a forward slash. Predictions were made for all steps, except for that of 6-phosphofructokinase, EC 2.7.1.11. be able to activate both formate and formaldehyde substrates by enzymes of tetrahydrofolate metabolism, converting them into the derivative N-5,10-methylenetetrahydrofolate (methyleneTHF) (4,16). The methyleneTHF enters the cyclic C-1 serine pathway by donating the C-1 moiety to combine with glycine producing serine, resulting ultimately in capture of carbon as acetyl-coenzyme A (CoA) and regeneration of the C-1 acceptor molecule glycine for the next cycle of capture.…”

QscR-Mediated Transcriptional Activation of Serine Cycle Genes in Methylobacterium extorquens AM1

“…Flux measurements also show that formate is produced in the H 4 MPT-linked pathway (28), and although most of it is oxidized via formate dehydrogenases, resulting in production of additional NADH (12), a small portion (12%) is converted into formyl-H 4 F, via the FtfL reaction (Fig. 5A) (18,21). FtfL is known to be present at high levels during growth on multicarbon substrates (21).…”

“…5A) (18,21). FtfL is known to be present at high levels during growth on multicarbon substrates (21). Under these conditions, formyl-H 4 F would build up, activating QscR via direct binding, and the activated QscR (QscR*) would then bind to the regulated promoters, resulting in increased transcription of both the serine cycle genes and the H 4 F-linked C 1 transfer pathway genes (Fig.…”

Methylamine Utilization via the N -Methylglutamate Pathway in Methylobacterium extorquens PA1 Involves a Novel Flow of Carbon through C 1 Assimilation and Dissimilation Pathways

“…Then, as observed during methanol growth in M. extorquens species (25), the tetrahydromethanopterin (H 4 MPT)-dependent formaldehyde oxidation pathway (26,27) would serve as the primary dissimilatory module. Formate, thus produced, would be incorporated into biomass through the tetrahydrofolate (H 4 F)-dependent C 1 assimilation pathway (28)(29)(30)(31) and the serine cycle (32, 33) (see Fig. S1 in the supplemental material).…”