Radical S-adenosyl-L-methionine (rSAM) enzymes bind one or more Fe-S clusters and catalyze transformations that produce complex and structurally diverse natural products. One of the clusters, a 4Fe-4S cluster, binds and reductively cleaves SAM to generate the 5\'-deoxyadenosyl radical, which initiates the catalytic cycle by H-atom transfer from the substrate. The role(s) of the additional auxiliary Fe-S clusters (ACs) remains largely enigmatic. The rSAM enzyme PapB catalyzes the formation of thioether cross-links between the β-carbon of an Asp and a Cys thiolate found in the PapA peptide. One of the two ACs in the protein binds to the substrate thiol where, upon formation of a thioether bond, one reducing equivalent is returned to the protein. However, for the next catalytic cycle to occur, the protein must undergo an electronic state isomerization, returning the electron to the SAM-binding cluster. Using a series of iron-sulfur cluster deletion mutants, our data support a model whereby the isomerization is an obligatorily intermolecular electron transfer event that can be mediated by redox active proteins or small molecules, likely via the second AC in PapB. Surprisingly, a mixture of FMN and NADPH is sufficient to support both the reductive and the isomerization steps. These findings lead to a new paradigm involving intermolecular electron transfer steps in the activation of rSAM enzymes that require multiple iron-sulfur clusters for turnover. The implications of these results for the biological activation of rSAM enzymes are discussed.

S-adenosyl-L-methionine (SAM) is the active form of methionine, which participates in various metabolic reactions and plays a vital role. It is mainly used as a precursor by three key metabolic pathways: trans-methylation, trans-sulfuration, and trans-aminopropylation. Methionine adenosyltransferase (MAT) is the only enzyme to produce SAM from methionine and ATP. However, there is no efficient and accurate method for high-throughput detection of SAM, which is the major obstacles of directed evolution campaigns for MAT. Herein, we established a colorimetric method for directed evolution of MAT based on detecting SAM by using glycine oxidase and glycine/sarcosine N-methyltransferase enzyme. Screening of MAT libraries revealed variant I303V/Q22R with 2.13-fold improved activity towards SAM in comparison to the wild type. Molecular dynamic simulation indicates that the loops more flexible and more conducive to SAM release.

The SpoU-TrmD (SPOUT) methyltransferase superfamily was designated when structural similarity was identified between the transfer RNA-modifying enzymes TrmH (SpoU) and TrmD. SPOUT methyltransferases are found in all domains of life and predominantly modify transfer RNA or ribosomal RNA substrates, though one instance of an enzyme with a protein substrate has been reported. Modifications placed by SPOUT methyltransferases play diverse roles in regulating cellular processes such as ensuring translational fidelity, altering RNA stability, and conferring bacterial resistance to antibiotics. This large collection of S-adenosyl-L-methionine-dependent methyltransferases is defined by a unique α/β fold with a deep trefoil knot in their catalytic (SPOUT) domain. Herein, we describe current knowledge of SPOUT enzyme structure, domain architecture, and key elements of catalytic function, including S-adenosyl-L-methionine co-substrate binding, beginning with a new sequence alignment that divides the SPOUT methyltransferase superfamily into four major clades. Finally, a major focus of this review will be on our growing understanding of how these diverse enzymes accomplish the molecular feat of specific substrate recognition and modification, as highlighted by recent advances in our knowledge of protein-RNA complex structures and the discovery of the dependence of one SPOUT methyltransferase on metal ion binding for catalysis. Considering the broad biological roles of RNA modifications, developing a deeper understanding of the process of substrate recognition by the SPOUT enzymes will be critical for defining many facets of fundamental RNA biology with implications for human disease.

Methionine restriction (MetR) can extend lifespan and delay the onset of aging-associated pathologies in most model organisms. Previously, we showed that supplementation with the metabolite S-adenosyl-L-homocysteine (SAH) extends lifespan and activates the energy sensor AMP-activated protein kinase (AMPK) in the budding yeast Saccharomyces cerevisiae. However, the mechanism involved and whether SAH can extend metazoan lifespan have remained unknown. Here, we show that SAH supplementation reduces Met levels and recapitulates many physiological and molecular effects of MetR. In yeast, SAH supplementation leads to inhibition of the target of rapamycin complex 1 (TORC1) and activation of autophagy. Furthermore, in Caenorhabditis elegans SAH treatment extends lifespan by activating AMPK and providing benefits of MetR. Therefore, we propose that SAH can be used as an intervention to lower intracellular Met and confer benefits of MetR.

The study of senescence preservative on cut flowers helps boost the commercial value of flowers. Senescence in cut flower is associated with an increase of ethylene production, and is significantly influenced by ethylene pathway. This study was conducted to investigate whether S-adenosyl-L-methionine (SAM) and aminocyclopropane-1-carboxylic acid (ACC) involved in the ethylene synthesis process are correlated with the lysosome. The alterations of lysosome which was treated with the ethylene precursors ACC and SAM in HeLa cell using the confocal laser scanning microscope were investigated. According to the experimental results, the activity of lysosomes increased concentration dependently by ACC treatment, however, no change was observed by SAM treatment. In addition, Liquid chromatography-mass spectrometry (LC/MS) analysis was performed to confirm the effect of lysosomal enzyme (LE) extracted from egg white on ACC reduction, but no change was observed. On the contrary, to confirm the effect of ACC on lysosomes, lysosomes were extracted from HeLa cells treated with 5 mM ACC and confirmed by FE-SEM. The results showed that the size of lysosomes treated with ACC is larger than that of the control, which was treated with distilled water. The lysosomes in the control group were distributed in various ranges from 0 to 800 nm, but those treated with 5 mM ACC were in the range of 400 nm to 800 nm or more. Therefore, lysosomes had no effect on ACC, the precursor of ethylene, the aging hormone of cut flowers, however, ACC had effect on lysosomes.

S-Adenosyl-L-methionine (SAM) plays important roles in trans-methylation, trans-sulfuration, and polyamine synthesis in all living cells, and it is also an effective cure for liver disease, depressive syndromes, and osteoarthritis. The increased demands of SAM in pharmaceuticals industry have aroused lots of attempts to improve its production. In this study, a multiple-copy integrative plasmid pYMIKP-SAM2 was introduced into the chromosome of wild-type Saccharomyces cerevisiae strain ZJU001 to construct the recombined strain R1-ZJU001. Further studies showed that the recombinant yeast exhibited higher enzymatic activity of methionine adenosyltransferase and improved its SAM biosynthesis. With a three-phase fed-batch strategy in 15-liter bench-top fermentor, 8.81 g/L SAM was achieved after 52 h cultivation of R1-ZJU001, about 27.1 % increase over its parent strain ZJU001, whereas the SAM content was also improved from 64.6 mg/g DCW to 91.0 mg/g DCW. Our results shall provide insights into the metabolic engineering of SAM pathway in yeast for improved productivity of SAM and subsequent industrial applications.