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

Yi-Ru Lee,Te-Sheng Lin,Nei-Li Chan,Shu-Jung Lai,Mei-Chin Lai,Mu-Sen Liu

doi:10.1038/srep38071

Yi-Ru Lee, Te-Sheng Lin + Show 4 more

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https://doi.org/10.1038/srep38071

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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.

Highlights

Phosphoethanolamine N-methyltransferase, norcoclaurine 6-O-methyltransferase, and demethylmycophenolic acid O-methyltransferase, respectively[16,17,18]
Consistent with previous sequence-based prediction[26,27], our structural analysis validated that MpGSMT belongs to the Class I methyltransferases, with a characteristic seven-stranded Rossmann-like α/βcatalytic core responsible for substrate and cofactor binding (Fig. 1a)
Similar to glycine N-methyltransferase (GNMT), the catalytic core of MpGSMT is further elaborated by a helical N-terminal region that precedes the β1 strand and a lid domain composed of a four-stranded antiparallel βsheet that inserts between the β5 strand and αEhelix (Fig. 1)

Summary

Introduction

Phosphoethanolamine N-methyltransferase, norcoclaurine 6-O-methyltransferase, and demethylmycophenolic acid O-methyltransferase, respectively[16,17,18]. We performed structural analysis on the glycine sarcosine N-methyltransferase (GSMT) from Methanohalophilus portucalensis strain FDF1T (MpGSMT) to understand how end product-mediated allosteric inhibition is achieved. Two dual-activity, SAM-dependent methyltransferases sharing an overlapping function are employed to carry out this three-step biosynthetic pathway as follows: MpGSMT adds the first and second methyl groups to glycine, and the sarcosine dimethylglycine N-methyltransferase (MpSDMT) catalyzes the second and third methylation events[20,22]. The pronounced sequence similarity between MpGSMT and GNMT agrees with their overlapping activity as follows: both enzymes catalyze the SAM-dependent methylation of glycine to produce sarcosine[24,25]. We report structural characterization of MpGSMT in a betaine-bound, catalytically inactivated state, which elucidates for the first time the structural basis by which the activity of a biosynthetic methyltransferase is regulated by end product-mediated feedback inhibition

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