γ-Glutamylmethylamide Is an Essential Intermediate in the Metabolism of Methylamine by Methylocella silvestris

Abstract

Methylocella silvestris BL2, a facultative methane utilizer, can grow on monomethylamine (MMA) as a sole carbon and nitrogen source. No activity of MMA dehydrogenase was detectable. Instead, this bacterium utilizes a methylated amino acid pathway (␥-glutamylmethylamide [GMA] and N-methylglutamate [NMG]) for MMA metabolism. The activities of the two key enzymes in this pathway, GMA synthetase and NMG dehydrogenase, were found when the bacterium was grown on MMA. GMA was detected by high-performance liquid chromatography-mass spectrometry only when the bacterium was grown on MMA but not when it was grown on methanol. Proteomic analysis of soluble and membrane fractions of the proteome from MMA-and methanolgrown cultures revealed that an eight-gene cluster (Msil2632 to Msil2639) was induced by MMA and cotranscribed as an operon, as shown by reverse transcription-PCR. GMA-dissimilating enzyme activity was also detected when it was grown on MMA. Formaldehyde and ammonium production from GMA was dependent on glutamate but not on ␣-ketoglutarate. Marker exchange mutagenesis of a putative GMAS gene homologue (gmas, Msil2635) within this eight-gene cluster, with a kanamycin gene cassette, abolished growth of M. silvestris on MMA as either a sole carbon or a sole nitrogen source. Overall, our results suggest that gmas is essential in MMA metabolism by M. silvestris.Monomethylamine (MMA) is ubiquitous in the environment. For example, putrefaction of proteins (14a, 17) and degradation of many nitrogen-containing pesticides and herbicides can release MMA (5, 16b, 18). In the marine environment, MMA is released from the degradation of quaternary amines, such as betaine, carnitine, choline, and trimethylamine N-oxide, which are used as osmolytes by many marine organisms (3, 6). Once released, MMA can be used by some microorganisms as a sole carbon and nitrogen source through different pathways. Methanogenic archaea, such as Methanosarcina and Methanomicrobium, can use MMA anaerobically as a substrate to produce methane via a methyltransferase system (28). Gram-positive bacteria, such as Arthrobacter, metabolize MMA aerobically via an oxidase, which breaks down MMA into formaldehyde and ammonium (39). Gram-negative bacteria such as Methylobacterium extorquens and Paracoccus denitrificans utilize MMA dehydrogenase, a multisubunit enzyme that generates formaldehyde and ammonium from MMA aerobically (9, 16). Many other Gram-negative bacteria, such as Aminobacter aminovorans (previously known as strain MA and strain MS), can use MMA as a sole carbon and nitrogen source aerobically; however, they lack MMA dehydrogenase. It has been shown that in these microorganisms, two unusual amino acids, ␥-glutamylmethylamide (GMA) and N-methylglutamate (NMG), are involved in MMA metabolism (1,23,33). In strain MA, an enzyme proposed as "NMG synthase" ("NMGS") converted MMA to NMG, which was subsequently oxidized to formaldehyde, regenerating glutamate (Glu) by a membrane-bounded particulate, NMG dehydrogenase (NMGDH) (33). The reactions carrie...

Bacterial flavin-containing monooxygenase is trimethylamine monooxygenase

“…We have previously shown that the former gene cluster was involved in MMA metabolism in M. silvestris, which encodes enzymes of the γ-glutamylmethylamide and N-methylglutamate pathway, converting MMA to formaldehyde (which is used as C and an energy source) and ammonium (which is used as an N source; Fig. 1B) (35).…”

“…M. silvestris was grown at 25°C either in a 4-L fermentor or in 125-mL serum vials using diluted mineral salt medium as described previously (35). To test if the trimethylamine monooxygenase (tmm) mutant could grow on methylated amines, growth experiments were set up in triplicate using 120-mL serum vials, containing 20 mL medium with an inoculum size of 10%.…”

“…To construct a tmm mutant of M. silvestris, a downstream region (containing a KpnI site) and an upstream region (containing an MluI site) of the target were amplified by PCR and subcloned into pGEM-T vector (Promega) together with a kanamycin gene cassette (amplified from pCM184) inserted between the two regions (primers used are listed in Table S1). The resulting plasmid was then cut with KpnI and MluI, and the 2.6-kb fragment containing the kanamycin gene cassette was electroporated into M. silvestris as described previously (35).…”

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

“…[13]), is a complex, multistep pathway in which the methyl group from MA is transferred to glutamate to form two novel amino acid derivatives, NMG and ␥-glutamylmethylamide (GMA), which are then oxidized to release formaldehyde (14). The NMG pathway involves at least three unique enzymes: Nmethylglutamate synthase (NMGS), N-methylglutamate dehydrogenase (NMGDH), and ␥-glutamylmethylamide synthetase (GMAS) (3,(15)(16)(17). While the enzymes involved in the NMG pathway were purified and characterized over 3 decades ago (18)(19)(20)(21)(22)(23), the genes were discovered very recently (3,15,17).…”

Complete Genome Sequence of the Chloromethane-Degrading Hyphomicrobium sp. Strain MC1

“…Genes encoding enzymes and proteins for oxidation of methanol (mxa) and methylamine (mgs and mgd [5,7] but not mau genes) were identified, together with proteins and enzymes involved in pyrroloquinoline quinone synthesis and tetrahydrofolate-and tetrahydromethanopterin-linked pathways. Genes for complete serine and ethylmalonyl coenzyme A pathways (1) for carbon assimilation were identified.…”