Crystal structures of γ-glutamylmethylamide synthetase provide insight into bacterial metabolism of oceanic monomethylamine

Monomethylamine (MMA) is an important climate-active oceanic trace gas and ubiquitous in the oceans. γ-Glutamylmethylamide synthetase (GmaS) catalyzes the conversion of MMA to γ-glutamylmethylamide, the first step in MMA metabolism in many marine bacteria. The gmaS gene occurs in ∼23% of microbial genomes in the surface ocean and is a validated biomarker to detect MMA-utilizing bacteria. However, the catalytic mechanism of GmaS has not been studied because of the lack of structural information. Here, the GmaS from Rhodovulum sp. 12E13 (RhGmaS) was characterized, and the crystal structures of apo-RhGmaS and RhGmaS with different ligands in five states were solved. Based on structural and biochemical analyses, the catalytic mechanism of RhGmaS was explained. ATP is first bound in RhGmaS, leading to a conformational change of a flexible loop (Lys287-Ile305), which is essential for the subsequent binding of glutamate. During the catalysis of RhGmaS, the residue Arg312 participates in polarizing the γ-phosphate of ATP and in stabilizing the γ-glutamyl phosphate intermediate; Asp177 is responsible for the deprotonation of MMA, assisting the attack of MMA on γ-glutamyl phosphate to produce a tetrahedral intermediate; and Glu186 acts as a catalytic base to abstract a proton from the tetrahedral intermediate to finally generate glutamylmethylamide. Sequence analysis suggested that the catalytic mechanism of RhGmaS proposed in this study has universal significance in bacteria containing GmaS. Our results provide novel insights into MMA metabolism, contributing to a better understanding of MMA catabolism in global carbon and nitrogen cycles.

[1]  Yung-Hun Yang,et al.  Production of L-theanine using Escherichia coli whole-cell overexpressing γ- glutamylmethylamide synthetase with baker's yeast. , 2020, Journal of microbiology and biotechnology.

[2]  S. Joye,et al.  Microbial metabolism of methanol and methylamine in the Gulf of Mexico: insight into marine carbon and nitrogen cycling , 2018, Environmental microbiology.

[3]  Kamini,et al.  Carbaryl as a Carbon and Nitrogen Source: an Inducible Methylamine Metabolic Pathway at the Biochemical and Molecular Levels in Pseudomonas sp. Strain C5pp , 2018, Applied and Environmental Microbiology.

[4]  L. Stein.  Methylamine: a vital nitrogen (and carbon) source for marine microbes. , 2017, Environmental microbiology.

[5]  N. Jehmlich,et al.  Methylamine as a nitrogen source for microorganisms from a coastal marine environment , 2017, Environmental microbiology.

[6]  D. Scanlan,et al.  Identification of dimethylamine monooxygenase in marine bacteria reveals a metabolic bottleneck in the methylated amine degradation pathway , 2017, The ISME Journal.

[7]  J. Murrell,et al.  Trimethylamine and trimethylamine N-oxide are supplementary energy sources for a marine heterotrophic bacterium: implications for marine carbon and nitrogen cycling , 2014, The ISME Journal.

[8]  J. Murrell,et al.  Bacterial metabolism of methylated amines and identification of novel methylotrophs in Movile Cave , 2014, The ISME Journal.

[9]  Merete Grung,et al.  Amines and amine-related compounds in surface waters: a review of sources, concentrations and aquatic toxicity. , 2014, The Science of the total environment.

[10]  Koichiro Tamura,et al.  MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. , 2013, Molecular biology and evolution.

[11]  M. Schumacher,et al.  Structures of the Bacillus subtilis Glutamine Synthetase Dodecamer Reveal Large Intersubunit Catalytic Conformational Changes Linked to a Unique Feedback Inhibition Mechanism* , 2013, The Journal of Biological Chemistry.

[12]  S. Archer,et al.  Ocean-atmosphere trace gas exchange. , 2012, Chemical Society reviews.

[13]  Yin Chen.  Comparative genomics of methylated amine utilization by marine Roseobacter clade bacteria and development of functional gene markers (tmm, gmaS). , 2012, Environmental microbiology.

[14]  J. Murrell,et al.  Bacterial flavin-containing monooxygenase is trimethylamine monooxygenase , 2011, Proceedings of the National Academy of Sciences.

[15]  H. Belrhali,et al.  Crystal structure of Type III glutamine synthetase: surprising reversal of the inter-ring interface. , 2011, Structure.

[16]  Randy J. Read,et al.  Overview of the CCP4 suite and current developments , 2011, Acta crystallographica. Section D, Biological crystallography.

[17]  J. Murrell,et al.  γ-Glutamylmethylamide Is an Essential Intermediate in the Metabolism of Methylamine by Methylocella silvestris , 2010, Applied and Environmental Microbiology.

[18]  J. Murrell,et al.  Monomethylamine as a Nitrogen Source for a Nonmethylotrophic Bacterium, Agrobacterium tumefaciens , 2010, Applied and Environmental Microbiology.

[19]  Vincent B. Chen,et al.  PHENIX: a comprehensive Python-based system for macromolecular structure solution , 2010, Acta crystallographica. Section D, Biological crystallography.

[20]  M. Kalyuzhnaya,et al.  Genetics of the glutamate‐mediated methylamine utilization pathway in the facultative methylotrophic beta‐proteobacterium Methyloversatilis universalis FAM5 , 2010, Molecular microbiology.

[21]  M. Wakayama,et al.  Cloning and Expression of Methylovorus mays No. 9 Gene Encoding γ-Glutamylmethylamide Synthetase: An Enzyme Usable in Theanine Formation by Coupling with the Alcoholic Fermentation System of Baker’s Yeast , 2008 .

[22]  M. Wakayama,et al.  Characterization of Theanine-Forming Enzyme from Methylovorus mays No. 9 in Respect to Utilization of Theanine Production , 2007, Bioscience, biotechnology, and biochemistry.

[23]  M. Kusunoki,et al.  Atomic Structure of Plant Glutamine Synthetase , 2006, Journal of Biological Chemistry.

[24]  Wladek Minor,et al.  HKL-3000: the integration of data reduction and structure solution--from diffraction images to an initial model in minutes. , 2006, Acta crystallographica. Section D, Biological crystallography.

[25]  S. Mowbray,et al.  Structure of Mycobacterium tuberculosis glutamine synthetase in complex with a transition-state mimic provides functional insights. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[26]  M. Simon,et al.  A newly discovered Roseobacter cluster in temperate and polar oceans , 2004, Nature.

[27]  D. Eisenberg,et al.  Structure-function relationships of glutamine synthetases. , 2000, Biochimica et biophysica acta.

[28]  P. Liss,et al.  Distributions and biogeochemistries of methylamines and ammonium in the Arabian Sea , 1999 .

[29]  M. Moran,et al.  Numerical dominance of a group of marine bacteria in the alpha-subclass of the class Proteobacteria in coastal seawater , 1997, Applied and environmental microbiology.

[30]  S. Liaw,et al.  Discovery of the ammonium substrate site on glutamine synthetase, A third cation binding site , 1995, Protein science : a publication of the Protein Society.

[31]  M. Lidstrom,et al.  Genetic organization of the mau gene cluster in Methylobacterium extorquens AM1: complete nucleotide sequence and generation and characteristics of mau mutants , 1994, Journal of bacteriology.

[32]  F. Robb,et al.  Evolutionary relationships of bacterial and archaeal glutamine synthetase genes , 1994, Journal of Molecular Evolution.

[33]  W. McIntire,et al.  Cloning, sequencing, expression, and regulation of the structural gene for the copper/topa quinone-containing methylamine oxidase from Arthrobacter strain P1, a gram-positive facultative methylotroph , 1993, Journal of bacteriology.

[34]  D. R. Woods,et al.  Recent developments on the regulation and structure of glutamine synthetase enzymes from selected bacterial groups. , 1993, FEMS microbiology reviews.

[35]  Y Tateno,et al.  Evolution of the glutamine synthetase gene, one of the oldest existing and functioning genes. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[36]  Y. Tonomura,et al.  Purification and Characterization of γ-Glutamylmethylamide Synthetase from Methylophaga sp. AA-30. , 1992, Bioscience, biotechnology, and biochemistry.

[37]  V. Davidson,et al.  Purification and properties of methylamine dehydrogenase from Paracoccus denitrificans , 1987, Journal of bacteriology.

[38]  T. Arndt.  Crystal , 2019, Springer Reference Medizin.

[39]  Karen N. Allen,et al.  research papers Acta Crystallographica Section D Biological , 2003 .

[40]  David Eisenberg,et al.  Novel subunit—subunit interactions in the structure of glutamine synthetase , 1986, Nature.