Late-stage methylation is a key technology in the development of pharmaceutical compounds. Methyltransferase biocatalysis may provide powerful options to insert methyl groups into complex molecules with high regio-and chemoselectivity. The challenge of a large-scale application of methyltransferases is their dependence on S-adenosylmethionine (SAM) as a stoichiometric, and thus exceedingly expensive co-substrate. As a solution to this problem, we and others have explored the use of methyl halides as reagents for the in situ regeneration of SAM. However, the need to handle volatile electrophiles, such as methyl iodide (MeI), may also hamper applications at scale. As a more practical solution, we have now developed an enzyme-catalyzed process for the regeneration of SAM with methyl toluene sulfonate. Herein, we describe enzymes from the thiopurine methyltransferase family that accept sulfate-and sulfonate-based methyl donors to convert Sadenosylhomocysteine into SAM with efficiencies that rival MeI-based reactions.

Ergothioneine is asulfur-containing histidine derivative that emerges from microbial biosynthesis and enters the human body through intestinal uptake and regulated distribution into specific tissues.A lthough the proteins involved in biosynthesis and uptake are well characterized,l ess is known aboutt he degradative pathways of ergothioneine. This report describes the crystal structure of the active form of ergothionasef rom the oral pathogen Treponemad enticola complexed with the substrate analogued esmethyl-ergothioneine sulfonica cid. This enzyme catalyzes the 1,2-elimination of trimethylamine from ergothioneine and ergothioneine sulfonic acid by using au nique mode of substrate activation combined with acid/base catalysis. This structural and mechanistic investigation revealed four essential catalytic residues, which are strictly conserved in homologous proteins from common gastrointestinal bacteria and numerous pathogenic bacteria, suggesting that bacterial activity mayp lay an important role in determining the availability of ergothioneine in healthy and diseased human tissue. Figure 3.To p: Proposed substrate binding mode and mechanism for substrate activationbyTdETL wt .Bottom:Chargec omplementary enzyme:ligand complexes that stabilize ap ositivec harge on the imidazole ring (green)a re more active that complexes with unbalanced chargeo ran eutral imidazole ring (red). Bold letters indicate the corresponding entries in Ta ble 1.

The following experimental techniques were used to determine the structure: X-RAY DIFFRACTIONThe reported resolution of this entry is 1.90 Å.Percentile scores (ranging between 0-100) for global validation metrics of the entry are shown in the following graphic. The table shows the number of entries on which the scores are based. MetricWhole archive (#Entries) Similar resolution (#Entries, resolution range(Å)) Clashscore 141614 6847 (1.90-1.90) Ramachandran outliers 138981 6760 (1.90-1.90) Sidechain outliers 138945 6760 (1.90-1.90) Full wwPDB X-ray Structure Validation Report 7NED2 Entry composition i

The substrate-bound formylglycine generating enzyme forms a trigonal planar tris-thiolate Cu(i) complex ready for oxygen activation.

Ergothioneine is an emergent factor in cellular redox biochemistry in humans and pathogenic bacteria. Broad consensus has formed around the idea that ergothioneine protects cells against reactive oxygen species. The recent discovery that anaerobic microorganisms make the same metabolite using oxygen-independent chemistry, indicates that ergothioneine also plays physiological roles under anoxic conditions. In this report we describe the crystal structure of the anaerobic ergothioneine biosynthetic enzyme EanB from the green sulfur bacterium Chlorobium limicola. This enzyme catalyzes oxidative sulfurization of Ntrimethyl histidine. Based on structural and kinetic evidence we describe the catalytic mechanism of this unusual C-S bond forming reaction. Significant active site conservation among distant EanB homologs suggests that oxidative sulfurization of heterocyclic substrates may occur in a broad range of bacteria.

Molecular oxygen (O2) is a sustainable oxidation reagent. O2 is strongly oxidizing but kinetically stable and its final reaction product is water. For these reasons learning how to activate O2 and how to steer its reactivity along desired reaction pathways is a longstanding challenge in chemical research.[1] Activation of ground‐state diradical O2 can occur either via conversion to singlet oxygen or by one‐electron reduction to superoxide. Many enzymes facilitate activation of O2 by direct fomation of a metal‐oxygen coordination complex concomitant with inner sphere electron transfer. The formylglycine generating enzyme (FGE) is an unusual mononuclear copper enzyme that appears to follow a different strategy. Atomic‐resolution crystal structures of the precatalytic complex of FGE demonstrate that this enzyme binds O2 juxtaposed, but not coordinated to the catalytic CuI. Isostructural complexes that contain AgI instead of CuI or nitric oxide instead of O2 confirm that formation of the initial oxygenated complex of FGE does not depend on redox activity. A stepwise mechanism that decouples binding and activation of O2 is unprecedented for metal‐dependent oxidases, but is reminiscent of flavin‐dependent enzymes.

Molecular oxygen (O2) is a sustainable oxidation reagent. O2 is strongly oxidizing but kinetically stable and its final reaction product is water. For these reasons learning how to activate O2 and how to steer its reactivity along desired reaction pathways is a longstanding challenge in chemical research.[1] Activation of ground‐state diradical O2 can occur either via conversion to singlet oxygen or by one‐electron reduction to superoxide. Many enzymes facilitate activation of O2 by direct fomation of a metal‐oxygen coordination complex concomitant with inner sphere electron transfer. The formylglycine generating enzyme (FGE) is an unusual mononuclear copper enzyme that appears to follow a different strategy. Atomic‐resolution crystal structures of the precatalytic complex of FGE demonstrate that this enzyme binds O2 juxtaposed, but not coordinated to the catalytic CuI. Isostructural complexes that contain AgI instead of CuI or nitric oxide instead of O2 confirm that formation of the initial oxygenated complex of FGE does not depend on redox activity. A stepwise mechanism that decouples binding and activation of O2 is unprecedented for metal‐dependent oxidases, but is reminiscent of flavin‐dependent enzymes.

Late-stage methylation is a key technology in the development of pharmaceutical compounds. Methyltransferase biocatalysis may provide powerful options to insert methyl groups into complex molecules with high regio-and chemoselectivity. The challenge of a large-scale application of methyltransferases is their dependence on S-adenosylmethionine (SAM) as a stoichiometric, and thus exceedingly expensive co-substrate. As a solution to this problem, we and others have explored the use of methyl halides as reagents for the in situ regeneration of SAM. However, the need to handle volatile electrophiles, such as methyl iodide (MeI), may also hamper applications at scale. As a more practical solution, we have now developed an enzyme-catalyzed process for the regeneration of SAM with methyl toluene sulfonate. Herein, we describe enzymes from the thiopurine methyltransferase family that accept sulfate-and sulfonate-based methyl donors to convert Sadenosylhomocysteine into SAM with efficiencies that rival MeI-based reactions.