Over the sixty years since Koshland initially formulated the classical mechanisms for retaining and inverting glycosidases, researchers have assembled a large body of supporting evidence and have documented variations of these mechanisms. Recently, however, researchers have uncovered a number of completely distinct mechanisms for enzymatic cleavage of glycosides involving elimination and/or hydration steps. In family GH4 and GH109 glycosidases, the reaction proceeds via transient NAD(+)-mediated oxidation at C3, thereby acidifying the proton at C2 and allowing for elimination across the C1-C2 bond. Subsequent Michael-type addition of water followed by reduction at C3 generates the hydrolyzed product. Enzymes employing this mechanism can hydrolyze thioglycosides as well as both anomers of activated substrates. Sialidases employ a conventional retaining mechanism in which a tyrosine functions as the nucleophile, but in some cases researchers have observed off-path elimination end products. These reactions occur via the normal covalent intermediate, but instead of an attack by water on the anomeric center, the catalytic acid/base residue abstracts an adjacent proton. These enzymes can also catalyze hydration of the enol ether via the reverse pathway. Reactions of α-(1,4)-glucan lyases also proceed through a covalent intermediate with subsequent abstraction of an adjacent proton to give elimination. However, in this case, the departing carboxylate "nucleophile" serves as the base in a concerted but asynchronous syn-elimination process. These enzymes perform only elimination reactions. Polysaccharide lyases, which act on uronic acid-containing substrates, also catalyze only elimination reactions. Substrate binding neutralizes the charge on the carboxylate, which allows for abstraction of the proton on C5 and leads to an elimination reaction via an E1cb mechanism. These enzymes can also cleave thioglycosides, albeit slowly. The unsaturated product of polysaccharide lyases can then serve as a substrate for a hydration reaction carried out by unsaturated glucuronyl hydrolases. This hydration is initiated by protonation at C4 and proceeds in a Markovnikov fashion rather than undergoing a Michael-type addition, giving a hemiketal at C5. This hemiketal then undergoes a rearrangement that results in cleavage of the anomeric bond. These enzymes can also hydrolyze thioglycosides efficiently and slowly turn over substrates with inverted anomeric configuration. The mechanisms discussed in this Account proceed through transition states that involve either positive or negative charges, unlike the exclusively cationic transition states of the classical Koshland retaining and inverting glycosidases. In addition, the distribution of this charge throughout the substrate can vary substantially. The nature of these mechanisms and their transition states means that any inhibitors or inactivators of these unusual enzymes probably differ from those presently used for Koshland retaining or inverting glycosidases.

Human pancreatic α-amylase (HPA) is responsible for degrading starch to malto-oligosaccharides, thence to glucose, and is therefore an attractive therapeutic target for the treatment of diabetes and obesity. Here we report the discovery of a unique lariat nonapeptide, by means of the RaPID (Random non-standard Peptides Integrated Discovery) system, composed of five amino acids in a head-to-side-chain thioether macrocycle and a further four amino acids in a 3 helical C terminus. This is a potent inhibitor of HPA (K = 7 nM) yet exhibits selectivity for the target over other glycosidases tested. Structural studies show that this nonapeptide forms a compact tertiary structure, and illustrate that a general inhibitory motif involving two phenolic groups is often accessed for tight binding of inhibitors to HPA. Furthermore, the work reported here demonstrates the potential of this methodology for the discovery of de novo peptide inhibitors against other glycosidases.

Unsaturated glucuronyl hydrolases (UGLs) from GH family 88 of the CAZy classification system cleave a terminal unsaturated sugar from the oligosaccharide products released by extracellular bacterial polysaccharide lyases. This pathway, which is involved in extracellular bacterial infection, has no equivalent in mammals. A novel mechanism for UGL has previously been proposed in which the enzyme catalyzes hydration of a vinyl ether group in the substrate, with subsequent rearrangements resulting in glycosidic bond cleavage. However, clear evidence for this mechanism has been lacking. In this study, analysis of the products of UGL-catalyzed reactions in water, deuterium oxide, and dilute methanol in water, in conjunction with the demonstration that UGL rapidly cleaves thioglycosides and glycosides of inverted anomeric configuration (substrates that are resistant to hydrolysis by classical glycosidases), provides strong support for this new mechanism. A hydration-initiated process is further supported by the observed UGL-catalyzed hydration of a C-glycoside substrate analogue. Finally, the observation of a small β-secondary kinetic isotope effect suggests a transition state with oxocarbenium ion character, in which the hydrogen at carbon 4 adopts an axial geometry. Taken together, these observations validate the novel vinyl ether hydration mechanism and are inconsistent with either inverting or retaining direct hydrolase mechanisms at carbon 1.

Background:The three-dimensional structures of ␤-glucuronidase have been solved only for the GH2 enzymes. Results: AcGlcA79A is composed of a (␤/␣) 8 -barrel domain and a ␤-domain. Conclusion:The substrate binding site of AcGlcA79A is adapted for recognition of GlcA as a substrate. Significance: This is the first report describing the crystal structure, mechanism, and catalytic residues of a GH79 enzyme.

Recent crystal structures of cysteine dioxygenase (CDO) suggest the presence of two posttranslational modifications adjacent to the catalytic iron center: a thioether cross-link between Cys93 and Tyr157 and extra electron density at Cys164 which was variously explained as cystine or cysteine sulfinic acid. Purification of recombinant rat CDO yields "mature" and "immature" forms with distinct electrophoretic mobilities. We have positively identified and characterized the two modifications in the products of three sequential proteolytic digestions using liquid chromatography coupled with tandem mass spectrometry. The cross-link is unique to the mature form and was identified in an ion of m/z 3,225.403, consistent with a Tyr-Cys cross-link of peptides Gly80-Phe94 with His155-Phe167. The cross-link is liable to cleavage by in-source decay and the resulting separate peptides were sequenced by collision-induced dissociation tandem mass spectrometry. Mass-spectrometric analysis of these same and overlapping peptides in the presence or absence of reductants and alkylating agents identified the second modification to be a cystine formed between Cys164 and exogenous cysteine as proposed earlier. Both modifications have been shown to form in the presence of high levels of cysteine and iron. This and the presence of small amounts of an apparently off-pathway cystine at position Cys93 suggest that although these conditions promote CDO maturation, they may actually arise via nonenzymatic, nonphysiological processes.

We report a strategy for efficient post-translational modification of a library of ribosomally-translated peptides by activation and elimination of cysteine to dehydroalanine then conjugate addition of a range of exogenous thiols, with an emphasis on carbohydrates.

De novo macrocyclic peptides, derived using selection technologies such as phage and mRNA display, present unique and unexpected solutions to challenging biological problems. This is due in part to their unusual folds, which are able to present side chains in ways not available to canonical structures such as α-helices and β-sheets. Despite much recent interest in these molecules, their folding and binding behavior remains poorly characterized. In this work, we present cocrystallization, docking, and solution NMR structures of three de novo macrocyclic peptides that all bind as competitive inhibitors with single-digit nanomolar K i to the active site of human pancreatic α-amylase. We show that a short stably folded motif in one of these is nucleated by internal hydrophobic interactions in an otherwise dynamic conformation in solution. Comparison of the solution structures with a target-bound structure from docking indicates that stabilization of the bound conformation is provided through interactions with the target protein after binding. These three structures also reveal a surprising functional convergence to present a motif of a single arginine sandwiched between two aromatic residues in the interactions of the peptide with the key catalytic residues of the enzyme, despite little to no other structural homology. Our results suggest that intramolecular hydrophobic interactions are important for priming binding of small macrocyclic peptides to their target and that high rigidity is not necessary for high affinity.

Nicotinamide N-methyltransferase (NNMT) methylates nicotinamide to form 1-methylnicotinamide (MNA) using S-adenosyl-L-methionine (SAM) as the methyl donor. The complexity of the role of NNMT in healthy and disease states is slowly...