Synthetic Reagents for Enzyme-Catalyzed Methylation.

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 S-adenosylhomocysteine into SAM with efficiencies that rival MeI-based reactions.

In Vitro Reconstitution of a Five-Step Pathway for Bacterial Ergothioneine Catabolism.

Ergothioneine is a histidine-derived sulfur metabolite that is biosynthesized by bacteria and fungi. Plants and animals absorb ergothioneine as a micronutrient from their environment or nutrition. Several different mechanisms of microbial ergothioneine production have been described in the past ten years. Much less is known about the genetic and structural basis for ergothioneine catabolism. In this report, we describe the in vitro reconstitution of a five-step pathway that degrades ergothioneine to l-glutamate, trimethylamine, hydrogen sulfide, carbon dioxide, and ammonia. The first two steps are catalyzed by the two enzymes ergothionase and thiourocanate hydratase. These enzymes are closely related to the first two enzymes in histidine catabolism. However, the crystal structure of thiourocanate hydratase from the firmicute Paenibacillus sp. reveals specific structural features that strictly differentiate the activity of this enzyme from that of urocanate hydratases. The final two steps are catalyzed by metal-dependent hydrolases that share most homology with the last two enzymes in uracil catabolism. The early and late part of this pathway are connected by an entirely new enzyme type that catalyzes desulfurization of a thiohydantoin intermediate. Homologous enzymes are encoded in many soil-dwelling firmicutes and proteobacteria, suggesting that bacterial activity may have a significant impact on the environmental availability of ergothioneine.

Non-Coordinative Binding of O2 at the Active Center of a Copper-Dependent Enzyme.

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.

Structure of formylglycine-generating enzyme in complex with copper and a substrate reveals an acidic pocket for binding and activation of molecular oxygen.

The formylglycine generating enzyme (FGE) catalyzes oxidative conversion of specific peptidyl-cysteine residues to formylglycine. FGE mediates O2-activation and hydrogen-atom abstraction in an active site that contains Cu(i) coordinated to two cysteine residues. Similar coordination geometries are common among copper-sensing transcription factors and copper-chaperone but are unprecedented among copper-dependent oxidases. To examine the mechanism of this unusual catalyst we determined the 1.04 Å structure of FGE from Thermomonospora curvata in complex with copper and a cysteine-containing peptide substrate. This structure unveils a network of four crystallographic waters and two active site residues that form a highly acidic O2-binding pocket juxtaposed to the trigonal planar tris-cysteine coordinated Cu(i) center. Comparison with structures of FGE in complex with Ag(i) and Cd(ii) combined with evidence from NMR spectroscopy and kinetic observations highlight several structural changes that are induced by substrate binding and prime the enzyme for O2-binding and subsequent activation.

Structure and Mechanism of Ergothionase from Treponema denticola.

Ergothioneine is a sulfur-containing histidine derivative that emerges from microbial biosynthesis and enters the human body through intestinal uptake and regulated distribution into specific tissues. Although the proteins involved in biosynthesis and uptake are well characterized, less is known about the degradative pathways of ergothioneine. This report describes the crystal structure of the active form of ergothionase from the oral pathogen Treponema denticola complexed with the substrate analogue desmethyl-ergothioneine sulfonic acid. This enzyme catalyzes the 1,2-elimination of trimethylamine from ergothioneine and ergothioneine sulfonic acid by using a unique 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 may play an important role in determining the availability of ergothioneine in healthy and diseased human tissue.

Structural and Mechanistic Basis for Anaerobic Ergothioneine Biosynthesis.

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 green sulfur bacterium Chlorobium limicola. This enzyme catalyzes the oxidative sulfurization of N-α-trimethyl histidine. On the basis of structural and kinetic evidence, we describe the catalytic mechanism of this unusual C-S bond-forming reaction. Significant active-site conservation among distant EanB homologues suggests that the oxidative sulfurization of heterocyclic substrates may occur in a broad range of bacteria.