Evolutionary advances are often fueled by unanticipated innovation. Directed evolution of a computationally designed enzyme suggests that dramatic molecular changes can also drive the optimization of primitive protein active sites. The specific activity of an artificial retro-aldolase was boosted >4,400 fold by random mutagenesis and screening, affording catalytic efficiencies approaching those of natural enzymes. However, structural and mechanistic studies reveal that the engineered catalytic apparatus, consisting of a reactive lysine and an ordered water molecule, was unexpectedly abandoned in favor of a new lysine residue in a substrate binding pocket created during the optimization process. Structures of the initial in silico design, a mechanistically promiscuous intermediate, and one of the most evolved variants highlight the importance of loop mobility and supporting functional groups in the emergence of the new catalytic center. Such internal competition between alternative reactive sites may have characterized the early evolution of many natural enzymes.

The complex plant flavonol glycoside montbretin A is a potent (Ki = 8 nM) and specific inhibitor of human pancreatic α-amylase with potential as a therapeutic for diabetes and obesity. Controlled degradation studies on montbretin A, coupled with inhibition analyses, identified an essential high-affinity core structure comprising the myricetin and caffeic acid moieties linked via a disaccharide. X-ray structural analyses of the montbretin A-human α-amylase complex confirmed the importance of this core structure and revealed a novel mode of glycosidase inhibition wherein internal π-stacking interactions between the myricetin and caffeic acid organize their ring hydroxyls for optimal hydrogen bonding to the α-amylase catalytic residues D197 and E233. This novel inhibitory motif can be reproduced in a greatly simplified analog, offering potential for new strategies for glycosidase inhibition and therapeutic development.

Trehalose synthase (TreS) catalyzes the reversible conversion of maltose into trehalose in mycobacteria as one of three biosynthetic pathways to this nonreducing disaccharide. Given the importance of trehalose to survival of mycobacteria, there has been considerable interest in understanding the enzymes involved in its production; indeed the structures of the key enzymes in the other two pathways have already been determined. Herein, we present the first structure of TreS from Mycobacterium smegmatis, thereby providing insights into the catalytic machinery involved in this intriguing intramolecular reaction. This structure, which is of interest both mechanistically and as a potential pharmaceutical target, reveals a narrow and enclosed active site pocket within which intramolecular substrate rearrangements can occur. We also present the structure of a complex of TreS with acarbose, revealing a hitherto unsuspected oligosaccharide-binding site within the C-terminal domain. This may well provide an anchor point for the association of TreS with glycogen, thereby enhancing its role in glycogen biosynthesis and degradation.

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.

As part of a search for selective, mechanism-based covalent inhibitors of human pancreatic a-amylase we describe the chemoenzymatic synthesis of the disaccharide analog a-glucosyl epi-cyclophellitol, demonstrate its stoichiometric reaction with human pancreatic a-amylase and evaluate the time dependence of its inhibition. X-ray crystallographic analysis of the covalent derivative so formed confirms its reaction at the active site with formation of a covalent bond to the catalytic nucleophile D197. The structure illuminates the interactions with the active site and confirms OH4' on the nonreducing end sugar as a good site for attachment of fluorescent tags in generating probes for localization and quantitation of amylase in vivo.Keywords: activity-based probes; amylase; conduritol epoxide; GH13 structure; glycosyl enzyme; mechanism-based inhibition Digestion of starch in mammals starts with endocleavage of its a-1,4-glucosidic bonds by salivary and pancreatic a-amylases. This liberates oligosaccharides that are hydrolyzed further by a-glucosidases located on the gut wall, thereby delivering glucose to the bloodstream. Control of blood glucose levels can be achieved clinically by a-glucosidase inhibitors (AGI) such as acarbose and miglitol, which inhibit the gut wall enzymes [1,2]. However unpleasant side effects, in part associated with passage of the oligosaccharides to the lower gut, can limit patient compliance. An alternative strategy, which our laboratory has been exploring, is to employ specific inhibitors of the a-amylases that allow dietary oligosaccharides such as sucrose to be processed normally. This approach should minimize side effects while still reducing blood glucose levels. To that end, we have taken two approaches. High-throughput screening of natural product extracts is one approach, and has led us to the potent (K i = 8.1 nM) human pancreatic a-amylase (HPA) inhibitor montbretin A, which controls blood glucose in diabetic animals, and for which we have determined three-dimensional structures of its complex with HPA [3,4].The other approach has been through design of mechanism-based inhibitors that form covalent adducts with the HPA active site. While 2-deoxy-2-Abbreviations CP, cyclophellitol; ECP, epi-cyclophellitol; G-ECP, glucosyl epi-cyclophellitol; HPA, human pancreatic a-amylase; LaMalP, Lactobacillus acidophilus NCFM maltose phosphorylase.