Structural Studies of the -Glycosidase from Sulfolobus solfataricus in Complex with Covalently and Noncovalently Bound Inhibitors † Tracey M. Gloster, ‡ Shirley Roberts, ‡ Vale ´rie M-A. Ducros, ‡ Giuseppe Perugino, ⊥ Mose ` Rossi, ⊥,§ Roland Hoos, | Marco Moracci, ⊥ Andrea Vasella, | and Gideon J. Davies* ,‡ Structural Biology Laboratory, Department of Chemistry, The UniVersity of York, Heslington, York YO10 5YW, United Kingdom, Institute of Protein BiochemistrysCNR, Via P. Castellino 111, 80131 Naples, Italy, Dipartimento di Chimica Biologica, UniVersita ` di Napoli “Federico II”, Via Mezzocannone 16, 80134 Naples, Italy, and Laboratorium fu ¨r Organische Chemie, HCI H 317, ETH-Ho ¨nggerberg, CH-8093 Zu ¨rich, Switzerland ReceiVed February 13, 2004; ReVised Manuscript ReceiVed March 18, 2004 ABSTRACT: Transition-state mimicry is increasingly important both to understand enzyme mechanism and to direct the synthesis of putative therapeutic agents. X-ray crystallography is able to provide vital information on the interactions between an enzyme and the potential inhibitor. Here we report the structures, at approximately 2 Å resolution, of a family GH1 -glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus, in complex with both covalently (derived from 2-fluoro-glycosides) and noncovalently (hydroximolactam) bound inhibitors. The enzyme has broad specificity, accommodating both gluco- and galacto-configured substrates, and the crystallographic data demonstrate that the only difference in the way these ligands bind lies in the interactions between Gln18, Glu432, and Trp433, and the hydroxyl group at the O3 and O4 positions. Inhibition by the differently configured ligands was also shown to be extremely similar, with K i values of 1.04 and 1.08 µM for the gluco and galacto epimers, respectively. The noncovalently bound inhibitors have a trigonal anomeric carbon, adopt a 4 H 3 (half- chair) conformation, and an interaction is formed between O2 and the catalytic nucleophile, all of which contribute to (partial) mimicry of the oxocarbenium-ion-like transition state. The inhibition of the -glycosidase from S. solfataricus by hydroximolactams is discussed in light of the emerging work on family GH1 glycosidase inhibition by a spectrum of putative transition-state mimics. Glycoside hydrolases have been shown, through the comparison of uncatalyzed (k uncat ) and catalyzed (k cat ) reaction rates under similar conditions, to accelerate the rate of glycosidic bond hydrolysis by a factor of approximately 10 17 , making them among the most proficient of enzymes (1). One may thus estimate (as described in ref 2), taking into account K M values around 10 -5 M, that the resultant dissociation constant of the enzyme-substrate (ES) complex at the transition state is around 10 -22 M(1). The high affinity for the transition state makes glycosidases particularly rewarding targets for inhibitor design through mimicry of this transition state. Indeed, a number of successful therapeutic agents, including Acarbose, Miglitol, N-butyl-deoxynojirimycin, Tamiflu and Relenza, have been targeted at this enzyme class (for example, see ref 3). Enzymes that catalyze the hydrolysis of glycosides have been classified into over 90 families on the basis of amino acid sequence similarities. 1 Within these families, “classical” hydrolysis of the glycosidic bond occurs via two mechanisms, which results in either net retention or inversion of config- uration at the anomeric carbon. An exo--glycosidase, Ss- Glc1, 2 from Sulfolobus solfataricus (an extremely thermo- acidophilic archaeon that optimally grows in hot springs at around 80 °C and pH 3), derived from the lacS gene, has been classified into glycoside hydrolase family GH1 (4). The three-dimensional structure of the native enzyme was first reported at 2.6 Å resolution (5). A large number of GH1 structures have now been reported; all are nonreducing-end specific exo-glycosidases with a wide variety of both glycon and aglycon specificities (for example, see refs 6-12). Most recently, the Thermotoga maritima GH1 enzyme was solved in complex with two imino-sugar inhibitors that, in harness with calorimetric measurements, provide provocative insight into enzyme inhibition (13). Family GH1 members perform catalysis with retention via a double-displacement mechanism through a covalent gly- cosyl-enzyme intermediate. During the first step of the canonical retention mechanism, the acid/base (invariably Asp or Glu) protonates the glycosidic oxygen to assist departure of the (poor) leaving group, with migration of the electro- † The work in Naples was supported by an Agenzia Spaziale Italiana project, contract no. I/R/365/02, and by a MIUR project, contract no. RBAU015B47, “Folding di proteine: l’altra meta ` del codice genetico”. The work in York was supported by the EPSRC and the BBSRC. G.J.D. is a Royal Society University Research fellow. * To whom correspondence should be addressed. Tel.: +44 1904 328260. Fax: +44 1904 328266. E-mail: davies@ysbl.york.ac.uk. ‡ The University of York. ⊥ Institute of Protein Biochemistry-CNR. § Universita ` di Napoli “Federico II”. | ETH-Ho ¨nggerberg. 1 http://afmb.cnrs-mrs.fr/∼cazy/CAZY/index.html. 2 Abbreviations: SS-Glc1, -glycosidase from Sulfolobus solfa- taricus; 2F-Glc, 2-deoxy-2-fluoro-glucose; 2F-Gal, 2-deoxy-2-fluoro- galactose; rms, root-mean-square; RMSD, root-mean-square deviation; GH1, glycoside hydrolase from family 1. 6101 Biochemistry 2004, 43, 6101-6109 10.1021/bi049666m CCC: $27.50 © 2004 American Chemical Society Published on Web 04/29/2004