Bacterial flavin-containing monooxygenase is trimethylamine monooxygenase

Abstract

Flavin-containing monooxygenases (FMOs) are one of the most important monooxygenase systems in Eukaryotes and have many important physiological functions. FMOs have also been found in bacteria; however, their physiological function is not known. Here, we report the identification and characterization of trimethylamine (TMA) monooxygenase, termed Tmm, from Methylocella silvestris, using a combination of proteomic, biochemical, and genetic approaches. This bacterial FMO contains the FMO sequence motif (FXGXXXHXXXF/Y) and typical flavin adenine dinucleotide and nicotinamide adenine dinucleotide phosphate-binding domains. The enzyme was highly expressed in TMA-grown M. silvestris and absent during growth on methanol. The gene, tmm, was expressed in Escherichia coli, and the purified recombinant protein had high Tmm activity. Mutagenesis of this gene abolished the ability of M. silvestris to grow on TMA as a sole carbon and energy source. Close homologs of tmm occur in many Alphaproteobacteria, in particular Rhodobacteraceae (marine Roseobacter clade, MRC) and the marine SAR11 clade (Pelagibacter ubique). We show that the ability of MRC to use TMA as a sole carbon and/or nitrogen source is directly linked to the presence of tmm in the genomes, and purified Tmm of MRC and SAR11 from recombinant E. coli showed Tmm activities. The tmm gene is highly abundant in the metagenomes of the Global Ocean Sampling expedition, and we estimate that 20% of the bacteria in the surface ocean contain tmm. Taken together, our results suggest that Tmm, a bacterial FMO, plays an important yet overlooked role in the global carbon and nitrogen cycles.

Fig. 1.

(A) A summary of comparative proteomics and transcriptional analyses of the three-gene cluster containing a bacterial FMO in M. silvestris. The function was based on the annotation from the genome sequence using BLASTP search; the abundance of each of the polypeptides is shown in percentages of the total soluble proteome in each condition, and the presence (+) or absence (−) of transcription of each gene was confirmed by RT-PCR. MeOH, methanol; MMA, monomethylamine; TMA, trimethylamine. (B) The proposed pathway for TMA oxidation involving Tmm.

Fig. 2.

A neighbor-joining phylogenetic tree showing the relationship of Tmm to other FMOs. The tree was drawn using the MEGA4 (50) based on an alignment of ∼450 amino acids of FMOs. Baeyer–Villiger monooxygenases were used as an outgroup. Bootstrap values of 100 replicates are shown.

Fig. 3.

Representative growth curves of Ruegeria pomeroyi DSS-3 and Roseovarius sp. 217 growing on TMA or ammonium as the sole nitrogen source. Controls were set up with no added nitrogen. (Insets) Activity of Tmm from the supernatant of the crude extract of R. pomeroyi DSS-3 and Roseovarius sp. 217 cultures, respectively. No enzyme activities were detected when ammonium was used as the sole nitrogen source. Error bars indicate SD of each experiment (n = 3).

Fig. 4.

An unrooted tree showing Tmm homologs retrieved from sequenced bacterial genomes and the Global Ocean Sampling expedition data set. The neighbor-joining tree was constructed using sequences retrieved from sequenced bacterial genomes (∼450 amino acids). Environmental sequences were added by parsimony. Bootstrap values were calculated based on 100 replicates. Homo sapiens FMO3 was used as the outgroup.