S-Adenosyl-L-cysteine

S-Adenosyl-L-methionine (AdoMet), S-adenosyl-L-homocysteine (L-AdoHcy), and related ribonucleosides have been oxidized with periodic acid to the corresponding 2',3'-dialdehydes. Both AdoMet dialdehyde and L-AdoHcy dialdehyde were observed to rapidly and irreversibly inactivate histamine N-methyltransferase (HMT). Equally active as an irreversible inhibitor was S-adenosyl-D-homocysteine dialdehyde (D-AdoHcy dialdehyde), which is consistent with the known affinity of HMT for S-adenosyl-D-homocysteine (D-AdoHcy). Other analogues of AdoHcy dialdehyde (S-adenosyl-L-cysteine dialdehyde, S-adenosyl-L-homocysteine sulfoxide dialdehyde, and adenosine dialdehyde) also produced irreversible inactivation of HMT, but at predictably slower rates. The corresponding acyclic 2',3'-ribonucleosides, which were obtained by NaBH4 reduction of the ribonucleosides dialdehydes, were found to be very weak, reversible inhibitors of HMT. Kinetic analysis of the inactivation of HMT produced by L-AdoHcy dialdehyde, AdoMet dialdehyde, and D-AdoHcy dialdehyde suggested mechanisms involving the formation of dissociable enzyme-inhibitor complexes prior to irreversible inactivation. Studies using L-[2,8-3H] AdoHcy dialdehyde revealed that incorporation of radioactivity into HMT closely paralleled the loss of enzyme activity. The results of these studies indicate that L-AdoHcy dialdehyde, D-AdoHcy dialdehyde, and AdoMet dialdehyde are affinity labeling reagents for HMT.

Caffeine (1,3,7-trimethylxanthine) is a secondary metabolite produced by certain plant species and an important component of coffee (Coffea arabica and Coffea canephora) and tea (Camellia sinensis). Here we describe the structures of two S-adenosyl-l-methionine-dependent N-methyltransferases that mediate caffeine biosynthesis in C. canephora 'robusta', xanthosine (XR) methyltransferase (XMT), and 1,7-dimethylxanthine methyltransferase (DXMT). Both were cocrystallized with the demethylated cofactor, S-adenosyl-L-cysteine, and substrate, either xanthosine or theobromine. Our structures reveal several elements that appear critical for substrate selectivity. Serine-316 in XMT appears central to the recognition of XR. Likewise, a change from glutamine-161 in XMT to histidine-160 in DXMT is likely to have catalytic consequences. A phenylalanine-266 to isoleucine-266 change in DXMT is also likely to be crucial for the discrimination between mono and dimethyl transferases in coffee. These key residues are probably functionally important and will guide future studies with implications for the biosynthesis of caffeine and its derivatives in plants.

Carbon-sulfur bond formation at aliphatic positions is a challenging reaction that is performed efficiently by radical S-adenosyl-L-methionine (SAM) enzymes. Here we report that 1,3-thiazolidines can act as ligands and substrates for the radical SAM enzyme HydE, which is involved in the assembly of the active site of [FeFe]-hydrogenase. Using X-ray crystallography, in vitro assays and NMR spectroscopy we identified a radical-based reaction mechanism that is best described as the formation of a C-centred radical that concomitantly attacks the sulfur atom of a thioether. To the best of our knowledge, this is the first example of a radical SAM enzyme that reacts directly on a sulfur atom instead of abstracting a hydrogen atom. Using theoretical calculations based on our high-resolution structures we followed the evolution of the electronic structure from SAM through to the formation of S-adenosyl-L-cysteine. Our results suggest that, at least in this case, the widely proposed and highly reactive 5'-deoxyadenosyl radical species that triggers the reaction in radical SAM enzymes is not an isolable intermediate.