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

Abstract

Monomethylamine (MMA) is an important climate-active oceanic trace gas and ubiquitous in the oceans. The γ-glutamylmethylamide synthetase (GmaS) catalyzes the conversion of MMA to γ-glutamylmethylamide (GMA), 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 due to 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 GMA. 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.

Discovery of the 1-naphthylamine biodegradation pathway reveals an enzyme that catalyzes 1-naphthylamine glutamylation

“…NpaA1 shows a similar architecture to GSI protein from Salmonella typhimurium ( St GS) with a 1.2 Å root mean square deviation (RMSD)(Gill & Eisenberg, 2001), and γ-glutamylmethylamide synthetase from Rhodovulum sp. 12E13 ( Rh GmaS) with a 1.0 Å RMSD for the aligned Cα coordinates (Wang et al, 2021). The coordinating residues involved in ATP, glutamate, and Mn 2+ binding are highly conserved between NpaA1 and GS (Figure 4—supplement figure 2A and 2B).…”

“…Docking analysis of NpaA1 and AtdA1 revealed V201 implicated in 1NA binding, in the NpaA1 pocket, leading to a larger naphthylamine-binding volume. In AtdA1, Y205 (conserved in GS proteins) with a larger tryptophan side-chain group may increase steric hindrance for naphthylamine’s diphenyl ring (Murray et al, 2013; Wang et al, 2021). Surprisingly, the V201Y mutant in NpaA1 exhibited higher catalytic activity for both 1NA and aniline compared to the wild-type (Figure 5B).…”

“…Similarly, GmaS, which possesses the 15 th motif, also acts on organic amines as substrates (Figure 3A). The 15 th motif is situated in the vicinity of the active pocket in GmaS (Wang et al, 2021) and GoaS, suggesting that this motif may play a role in facilitating the binding of organic compounds in these GS-like enzymes. Figure 5-Source data1.…”

Characterization of the Trimethylamine N-Oxide Transporter From Pelagibacter Strain HTCC1062 Reveals Its Oligotrophic Niche Adaption

“…Then, TMAO in the cells is catabolized to DMA by TMAO demethylase Tdm ( Lidbury et al, 2014 ; Sun et al, 2019 ). With the catalysis of DMA monooxygenase DmmDABC, DMA is further catabolized to MMA ( Lidbury et al, 2017 ; Sun et al, 2019 ), which can be converted to γ-glutamylmethylamide by γ-glutamylmethylamide synthetase GmaS ( Chen Y. et al, 2010 ; Wischer et al, 2015 ; Wang et al, 2021 ). Bioinformatic analysis indicated that tmoXWV homologs are prevalent in SAR11 bacteria ( Lidbury et al, 2014 ).…”