Cloning and Expression of<i>Methylovorus mays</i>No. 9 Gene Encoding γ-Glutamylmethylamide Synthetase: An Enzyme Usable in Theanine Formation by Coupling with the Alcoholic Fermentation System of Baker’s Yeast

Yamamoto, Sachiko; Wakayama, Mamoru; Tachiki, Takashi

doi:10.1271/bbb.70462

Cited by 30 publications

(25 citation statements)

References 35 publications

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“…ORF Msil2635 encodes a glutamine synthetase-like protein; however, sequence analysis indicated that this gene (26). This gene also shows 41% identity to a newly characterized GMAS from Methylovorus mays (38) (Fig. 3).…”

Section: Resultsmentioning

confidence: 94%

“…The synthesis of GMA was proposed to be carried out by a glutamine synthetase-like protein, and the reaction was dependent on ATP and Mg 2ϩ . However, the fate of GMA in such a GMA-dependent MMA pathway is not clear.Although enzymes in these methylated amine-mediated pathways have been purified earlier, such as "NMGS" (31), NMGDH (2), and GMAS (21, 25, 37), the genes involved in these pathways have been studied only recently (24,38).Methylocella silvestris BL2 is a facultative methane-oxidizing bacterium belonging to the Alphaproteobacteria (11,13,35). It can grow on methylamine as a sole carbon and nitrogen source.…”

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confidence: 99%

“…However, the fate of GMA in such a GMA-dependent MMA pathway is not clear. Although enzymes in these methylated amine-mediated pathways have been purified earlier, such as "NMGS" (31), NMGDH (2), and GMAS (21,25,37), the genes involved in these pathways have been studied only recently (24,38).…”

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confidence: 99%

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

Chen

Scanlan

Song

et al. 2010

Appl Environ Microbiol

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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...

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Section: Resultsmentioning

confidence: 94%

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confidence: 99%

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

Chen

Scanlan

Song

et al. 2010

Appl Environ Microbiol

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“…9) was prepared by a method described previously. 22) One unit of GMAS was defined as the amount of enzyme forming 1 mmol ofglutamylhydroxamate per min at 30 C in a reaction mixture consisting of 50 mM sodium glutamate, 15 mM hydroxylamine, 7.5 mM ATP, 30 mM MgCl 2 , 100 mM imidazole buffer (pH 8.0), and recombinant GMAS.…”

Section: Methodsmentioning

confidence: 99%

The Involvement of L-Type Amino Acid Transporters in Theanine Transport

Yamamoto

Kimura

Tachiki

et al. 2012

Bioscience, Biotechnology, and Biochemistry

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“…19,20) The amino acid sequence of the latter enzyme showed significant identity to GS. 21) -Glutamylcysteine synthetase (GCS, EC 6.3.2.2) catalyzes the ATP-dependent synthesis of -glutamylcysteine from Glu and Cys, the first step in the reaction of glutathione formation. 22,23) The gshA gene encoding GCS has also been identified in a variety of organism, including E. coli.…”

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confidence: 99%

A Novel Catalytic Ability of γ-Glutamylcysteine Synthetase ofEscherichia coliand Its Application in Theanine Production

Miyake

Kakita

2009

Bioscience, Biotechnology, and Biochemistry

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-Glutamylcysteine synthetase (GCS, EC 6.3.2.2) catalyzes the formation of -glutamylcysteine from L-glutamic acid (Glu) and L-cysteine (Cys) in an ATPdependent manner. While GCS can use various amino acids as substrate, little is known about whether it can use non-amino acid compounds in place of Cys. We determined that GCS from Escherichia coli has the ability to combine Glu and amines to form -glutamylamides. The reaction rate depended on the length of the methylene chain of the amines in the following order: n-propylamine > butylamine > ethylamine methylamine. The optimal pH for the reaction was narrower and more alkaline than for the reaction with an amino acid. The newly found catalytic ability of GCS was used in the production of theanine (-glutamylethylamine). The resting cells of E. coli expressing GCS, in which ATP was regenerated through glycolysis, synthesized 12.1 mM theanine (18 h) from 429 mM ethylamine.

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Cloning and Expression ofMethylovorus maysNo. 9 Gene Encoding γ-Glutamylmethylamide Synthetase: An Enzyme Usable in Theanine Formation by Coupling with the Alcoholic Fermentation System of Baker’s Yeast

Cited by 30 publications

References 35 publications

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

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

The Involvement of L-Type Amino Acid Transporters in Theanine Transport

A Novel Catalytic Ability of γ-Glutamylcysteine Synthetase ofEscherichia coliand Its Application in Theanine Production

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