The unrelated active sites of Ni-Fe and Fe-only hydrogenases have several common features: coordination of diatomic ligands to an Fe ion; a vacant coordination site on one of the metal ions representing a possible substrate-binding site; a thiolate-bridged binuclear center; and plausible proton- and electron-transfer pathways and substrate channels. The diatomic coordination to Fe ions makes them low spin and favors low redox states, which may be required for catalysis. Complex electron paramagnetic resonance signals typical of Fe-only hydrogenases arise from magnetic interactions between the [4Fe-4S] cluster and the active site binuclear center. The paucity of protein ligands to this center suggests that it was imported from the inorganic world as an already functional unit.

Fe-only hydrogenases, as well as their NiFe counterparts, display unusual intrinsic high-frequency IR bands that have been assigned to CO and CN(-) ligation to iron in their active sites. FTIR experiments performed on the Fe-only hydrogenase from Desulfovibrio desulfuricans indicate that upon reduction of the active oxidized form, there is a major shift of one of these bands that is provoked, most likely, by the change of a CO ligand from a bridging position to a terminal one. Indeed, the crystal structure of the reduced active site of this enzyme shows that the previously bridging CO is now terminally bound to the iron ion that most likely corresponds to the primary hydrogen binding site (Fe2). The CO binding change may result from changes in the coordination sphere of Fe2 or its reduction. Superposition of this reduced active site with the equivalent region of a NiFe hydrogenase shows a remarkable coincidence between the coordination of Fe2 and that of the Fe ion in the NiFe cluster. Both stereochemical and mechanistic considerations suggest that the small organic molecule found at the Fe-only hydrogenase active site and previously modeled as 1,3-propanedithiolate may, in fact, be di-(thiomethyl)-amine.

4286 6.1. Oxidized Inactive States of the Ni−Fe Site and Radiation Effects 4286 6.2. Oxidative Damage of FeS Clusters in [FeFe]-Hydrogenases 4288 6.3. Hydrophobic Tunnels in [NiFe]-Hydrogenases 4288 6.4. Hydrophobic Tunnels in [FeFe]-Hydrogenase 4291 6.5. Hydrogen Sensors Related to [NiFe]-Hydrogenases 4292 6.6. Oxygen-Insensitive [NiFe]-Hydrogenases from Ralstonia eutropha 4294 7. Evolutionary Relationships of Hydrogenases to Other Proteins 4294 7.1. Comparison of [NiFe]-Hydrogenase with Complex I 4294 7.2. Comparison of [FeFe]-Hydrogenase to Narf-like Proteins 4296 8. Substrate Binding and Catalysis 4297 8.1. [NiFe]-Hydrogenases 4297 8.2. [FeFe]-Hydrogenases 4297 8.3. Comparison of Active Redox States in [NiFe]and [FeFe]-Hydrogenases 4299 9. Concluding Remarks 4299 10. Acknowledgments 4300 11. Note Added after ASAP Publication 4300 12. References 4300

This review provides a comprehensive update of the advances in discovery, biosynthesis, and engineering of ribosomally-synthesized and post-translationally modified peptides (RiPPs).

Cholinesterases are among the most efficient enzymes known. They are divided into two groups: acetylcholinesterase, involved in the hydrolysis of the neurotransmitter acetylcholine, and butyrylcholinesterase of unknown function. Several crystal structures of the former have shown that the active site is located at the bottom of a deep and narrow gorge, raising the question of how substrate and products enter and leave. Human butyrylcholinesterase (BChE) has attracted attention because it can hydrolyze toxic esters such as cocaine or scavenge organophosphorus pesticides and nerve agents. Here we report the crystal structures of several recombinant truncated human BChE complexes and conjugates and provide a description for mechanistically relevant non-productive substrate and product binding. As expected, the structure of BChE is similar to a previously published theoretical model of this enzyme and to the structure of Torpedo acetylcholinesterase. The main difference between the experimentally determined BChE structure and its model is found at the acyl binding pocket that is significantly bigger than expected. An electron density peak close to the catalytic Ser 198 has been modeled as bound butyrate.

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The crystal structure of biotin synthase from Escherichia coli in complex with S-adenosyl-Lmethionine and dethiobiotin has been determined to 3.4 angstrom resolution. This structure addresses how "AdoMet radical" or "radical SAM" enzymes use Fe 4 S 4 clusters and S-adenosyl-L-methionine to generate organic radicals. Biotin synthase catalyzes the radical-mediated insertion of sulfur into dethiobiotin to form biotin. The structure places the substrates between the Fe 4 S 4 cluster, essential for radical generation, and the Fe 2 S 2 cluster, postulated to be the source of sulfur, with both clusters in unprecedented coordination environments.Biotin synthase (BioB) catalyzes the final step in the biotin biosynthetic pathway, the conversion of dethiobiotin (DTB) to biotin. This remarkable reaction uses organic radical chemistry for the insertion of a sulfur atom between nonactivated carbons C6 and C9 of DTB (Scheme 1). BioB is a member of the "AdoMet radical" or "radical SAM" superfamily, which is characterized by the presence of a conserved CxxxCxxC sequence motif (C, Cys; x, any amino acid) that coordinates an essential Fe 4 S 4 cluster, as well as by the use of S-adenosyl-Lmethionine (AdoMet or SAM) for radical generation (1-3). AdoMet radical enzymes act on a wide variety of biomolecules. For example, BioB and lipoyl-acyl carrier protein synthase (LipA) are involved in vitamin biosynthesis; lysine 2,3-aminomutase (LAM) facilitates the fermentation of lysine; class III ribonucleotide reductase (RNR) activase and pyruvate formatelyase (PFL) activase catalyze the formation of glycyl radicals in their respective target proteins; and spore photoproduct lyase repairs ultraviolet light-induced DNA damage.AdoMet has been referred to as the "poor man's adenosylcobalamin" (4) because of the ability of both cofactors to generate a highly reactive 5′-deoxyadenosyl radical (5′-dA·), formed through homolytic cleavage of a C-Co bond in the case of adenosylcobalamin (AdoCbl) and through reductive cleavage of a C-S bond in the case of AdoMet (5). In AdoMet radical enzymes, the formation of 5′-dA· requires the addition of one electron, provided in E. coli by reduced flavodoxin and transferred first into an Fe 4 S 4 cluster and then into AdoMet (3). In the reaction catalyzed by BioB, there is general agreement that 5′-dA· generated from AdoMet oxidizes DTB (6), but the number and types of FeS clusters and of other cofactors involved in the reaction have been a subject of controversy (7)(8)(9)(10)(11)(12)(13)(14). Protein preparation-dependent cofactor differences have led to two mechanistic proposals for the method of S insertion in BioB. One proposal involves the use of an Fe 2 S 2 cluster as the sulfur source for biotin, and is consistent with 34 S isotopic labeling studies (15) and with the observed destruction of an Fe 2 S 2 cluster

The multifunctional nature of Alzheimer's disease calls for MTDLs (multitarget-directed ligands) to act on different components of the pathology, like the cholinergic dysfunction and amyloid aggregation. Such MTDLs are usually on the basis of cholinesterase inhibitors (e.g. tacrine or huprine) coupled with another active molecule aimed at a different target. To aid in the design of these MTDLs, we report the crystal structures of hAChE (human acetylcholinesterase) in complex with FAS-2 (fasciculin 2) and a hydroxylated derivative of huprine (huprine W), and of hBChE (human butyrylcholinesterase) in complex with tacrine. Huprine W in hAChE and tacrine in hBChE reside in strikingly similar positions highlighting the conservation of key interactions, namely, π-π/cation-π interactions with Trp86 (Trp82), and hydrogen bonding with the main chain carbonyl of the catalytic histidine residue. Huprine W forms additional interactions with hAChE, which explains its superior affinity: the isoquinoline moiety is associated with a group of aromatic residues (Tyr337, Phe338 and Phe295 not present in hBChE) in addition to Trp86; the hydroxyl group is hydrogen bonded to both the catalytic serine residue and residues in the oxyanion hole; and the chlorine substituent is nested in a hydrophobic pocket interacting strongly with Trp439. There is no pocket in hBChE that is able to accommodate the chlorine substituent.