Structural Analysis of Glycine Sarcosine N-methyltransferase from Methanohalophilus portucalensis Reveals Mechanistic Insights into the Regulation of Methyltransferase Activity

Abstract

Methyltransferases play crucial roles in many cellular processes, and various regulatory mechanisms have evolved to control their activities. For methyltransferases involved in biosynthetic pathways, regulation via feedback inhibition is a commonly employed strategy to prevent excessive accumulation of the pathways’ end products. To date, no biosynthetic methyltransferases have been characterized by X-ray crystallography in complex with their corresponding end product. Here, we report the crystal structures of the glycine sarcosine N-methyltransferase from the halophilic archaeon Methanohalophilus portucalensis (MpGSMT), which represents the first structural elucidation of the GSMT methyltransferase family. As the first enzyme in the biosynthetic pathway of the osmoprotectant betaine, MpGSMT catalyzes N-methylation of glycine and sarcosine, and its activity is feedback-inhibited by the end product betaine. A structural analysis revealed that, despite the simultaneous presence of both substrate (sarcosine) and cofactor (S-adenosyl-L-homocysteine; SAH), the enzyme was likely crystallized in an inactive conformation, as additional structural changes are required to complete the active site assembly. Consistent with this interpretation, the bound SAH can be replaced by the methyl donor S-adenosyl-L-methionine without triggering the methylation reaction. Furthermore, the observed conformational state was found to harbor a betaine-binding site, suggesting that betaine may inhibit MpGSMT activity by trapping the enzyme in an inactive form. This work implicates a structural basis by which feedback inhibition of biosynthetic methyltransferases may be achieved.

Identification, structure and function of the methyltransferase involved in the biosynthesis of the dithiolopyrrolone antibiotic xenorhabdin

“…Crystallization trials with the pure protein ( Figure S13a, b ) yielded diffraction- quality crystals that allowed us to solve the structure of XrdM (residues 4-246) in complex with the cofactor S-adenosylhomocysteine (SAH) to 2.1 Å resolution ( Table S9 ) using the AlphaFold2 prediction 21 as a molecular replacement model ( Figure S14a ). The structure of XrdM is similar to many other MTs ( Table S10 ), but most related to ToxA, a dual-specificity N -MT that catalyzes the last two steps of toxoflavin biosynthesis 22 , as well as to glycine sarcosine N -MT 23 . XrdM folds into two subdomains at the interface of which a pronounced cofactor- and substrate-binding cleft is located ( Figure 4a ).…”

A simplified characterization of S-adenosyl-l-methionine-consuming enzymes with 1-Step EZ-MTase: a universal and straightforward coupled-assay for in vitro and in vivo setting

“… 54 However, we successfully superimposed this structure (PDB: ; 2O57) 54 with the SAH·sarcosine complex of the glycine sarcosine N -methyltransferase from Methanohalophilus portucalense ( Mp GSMT; PDB: ; 5HIL). 61 Both structures display a good alignment of their N -terminus (Fig. S6; † light shades) and the cofactor binding site was easily identified within this conserved domain.…”

Improving Microbial Cell Factory Performance by Engineering SAM Availability

“…SAM serves as the methyl donor (Figure ). So far, this pathway is only found in a few microorganisms, such as the halophilic and halotolerant species (Thioalkalivibrio versutus, Methanohalophilus portucalensis, and Aphanothece halophytica ). Because the methylation pathway is SAM consuming, the oxidative pathway is more suitable for enhancing betaine de novo biosynthesis.…”