TY - JOUR A1 - van Aalten, Daan M. F. A1 - Komander, David A1 - Synstad, Bjoenar A1 - Gaseidnes, Sigrid A1 - Peter, Martin G. A1 - Eijsink, Vincent G. H. T1 - Structural Insights into the catalytic mechanism of a family 18 exochitinase N2 - Chitinase B (ChiB) from Serratia marcescens is a family 18 exochitinase whose catalytic domain has a TIM-barrel fold with a tunnel-shaped active site. We have solved structures of three ChiB complexes that reveal details of substrate binding, substrateassisted catalysis, and product displacement. The structure of an inactive ChiB mutant (E144Q) complexed with a pentameric substrate (binding in subsites 22 to 13) shows closure of the ''roof'' of the active site tunnel. It also shows that the sugar in the 21 position is distorted to a boat conformation, thus providing structural evidence in support of a previously proposed catalytic mechanism. The structures of the active enzyme complexed to Allosamidin (an analogue of a proposed reaction intermediate) and of the active enzyme soaked with pentameric substrate show events after cleavage of the glycosidic bond. The latter structure shows reopening of the roof of the active site tunnel and enzyme-assisted product displacement in the 11 and 12 sites, allowing a water molecule to approach the reaction center. Catalysis is accompanied by correlated structural changes in the core of the TIM barrel that involve conserved polar residues whose functions were hitherto unknown. These changes simultaneously contribute to stabilization of the reaction intermediate and alternation of the pKa of the catalytic acid during the catalytic cycle. Y1 - 2002 ER - TY - JOUR A1 - Vaaje-Kolstad, G. A1 - Vasella, A. A1 - Peter, Martin G. A1 - Netter, C. A1 - Houston, Douglas R. A1 - Westereng, B. A1 - Synstad, Bjoenar A1 - Eijsink, Vincent G. H. A1 - van Aalten, Daan M. F. T1 - Interactions of a family 18 chitinase with the designed inhibitor HM508 and its degradation product, chitobiono- delta-lactone N2 - We describe enzymological and structural analyses of the interaction between the family 18 chitinase ChiB from Serratia marcescens and the designed inhibitor N,N'-diacetylchitobionoxime-N-phenylcarbamate (HM508). HM508 acts as a competitive inhibitor of this enzyme with a K-i in the 50 muM range. Active site mutants of ChiB show K-i values ranging from 1 to 200 muM, providing insight into some of the interactions that determine inhibitor affinity. Interestingly, the wild type enzyme slowly degrades HM508, but the inhibitor is essentially stable in the presence of the moderately active D142N mutant of ChiB. The crystal structure of the D142N-HM508 complex revealed that the two sugar moieties bind to the -2 and -1 subsites, whereas the phenyl group interacts with aromatic side chains that line the +1 and +2 subsites. Enzymatic degradation of HM508, as well as a Trp-->Ala mutation in the +2 subsite of ChiB, led to reduced affinity for the inhibitor, showing that interactions between the phenyl group and the enzyme contribute to binding. Interestingly, a complex of enzymatically degraded HM508 with the wild type enzyme showed a chitobiono-delta- lactone bound in the -2 and -1 subsites, despite the fact that the equilibrium between the lactone and the hydroxy acid forms in solution lies far toward the latter. This shows that the active site preferentially binds the E-4 conformation of the -1 sugar, which resembles the proposed transition state of the reaction Y1 - 2004 SN - 0021-9258 ER - TY - JOUR A1 - Vaaje-Kolstad, G. A1 - Houston, Douglas R. A1 - Rao, F. V. A1 - Peter, Martin G. A1 - Synstad, Bjoenar A1 - van Aalten, Daan M. F. A1 - Eijsink, Vincent G. H. T1 - Structure of the D142N mutant of the family 18 chitinase ChiB from Serratia marcescens and its complex with allosamidin N2 - Catalysis by ChiB, a family 18 chitinase from Serratia marcescens, involves a conformational change of Asp142 which is part of a characteristic D140XD142XE144 sequence motif In the free enzyme Asp142 points towards Asp140, whereas it rotates towards the catalytic acid, Glu144, upon ligand binding. Mutation of Asp142 to Asn reduced k(cat) and affinity for allosamidin, a competitive inhibitor. The X-ray structure of the D142N mutant showed that Asn142 points towards Glu144 in the absence of a ligand. The active site also showed other structural adjustments (Tyr10, Ser93) that had previously been observed in the wild-type enzyme upon substrate binding. The X-ray structure of a complex of D142N with allosamidin, a pseudotrisaccharide competitive inhibitor, was essentially identical to that of the wild-type enzyme in complex with the same compound. Thus, the reduced allosamidin affinity in the mutant is not caused by structural changes but solely by the loss of electrostatic interactions with Asp142. The importance of electrostatics was further confirmed by the pH dependence of catalysis and allosamidin inhibition. The pH-dependent apparent affinities for allosamidin were not correlated with k(cat), indicating that it is probably better to view the inhibitor as a mimic of the oxazolinium ion reaction intermediate than as a transition state analogue. (C) 2003 Elsevier B.V. All rights reserved Y1 - 2004 SN - 1570-9639 ER - TY - JOUR A1 - Thiele, Gabriela A1 - Rottmann, Antje A1 - Germer, Antje A1 - Kleinpeter, Erich A1 - Spindler, Klaus-Dieter A1 - Synstad, Bjoenar A1 - Eijsink, Vincent G. H. A1 - Peter, Martin G. T1 - Synthesis and conformational analysis of pseudosugar analogues of chitotriose N2 - In this article, the synthesis of analogs of N,N',N''-triacetylchitotriose in which the central sugar residue was replaced by a succinic acid is presented. Mol. modeling calcns. revealed that the pseudotrisaccharides exist in low energy extended conformations which show similar space filling as N,N',N''-triacetylchitotriose. Of the N,N',N''-triacetylchitotriose pseudosugar analogs tested as chitinase inhibitors, none showed any appreciable competition (numerical data not presented). The conformational anal. along with further synthetic efforts will hopefully lead to more efficient pseudosaccharides as chitinase inhibitors. Y1 - 2002 ER - TY - JOUR A1 - Rottmann, Antje A1 - Synstad, Bjoenar A1 - Thiele, G. A1 - Schanzenbach, Dirk A1 - Eijsink, Vincent G. H. A1 - Peter, Martin G. T1 - Approaches towards the design of new chitinase inhibitors Y1 - 1999 SN - 3-9806494-5-8 ER - TY - JOUR A1 - Rottmann, Antje A1 - Synstad, Bjoenar A1 - Eijsink, Vincent G. H. A1 - Peter, Martin G. T1 - Synthesis of N-acetylglucosaminyl and diacetylchitobiosyl amides of heterocyclic carboxylic acids as potential chitinase inhibitors Y1 - 1999 ER - TY - JOUR A1 - Letzel, Matthias C. A1 - Synstad, Bjoenar A1 - Eijsink, Vincent G. H. A1 - Peter-Katalinic, Jasna A1 - Peter, Martin G. T1 - Libraries of chito-oligosaccharides of mixed acetylation patterns and their interactions with chitinases Y1 - 1999 SN - 3-9806494-5-8 ER - TY - JOUR A1 - Houston, Douglas R. A1 - Shiomi, Kazuro A1 - Arai, Noriko A1 - Omura, Satoshi A1 - Peter, Martin G. A1 - Turberg, Andreas A1 - Synstad, Bjoenar A1 - Eijsink, Vincent G. H. A1 - Van Aalten, Daan M. F. T1 - High-resolution structures of a chitinase complexed with natural product cyclopentapeptide inhibitors : mimicry of carbohydrate substrate N2 - Over the past years, family 18 chitinases have been validated as potential targets for the design of drugs against human pathogens that contain or interact with chitin during their normal life cycles. Thus far, only one potent chitinase inhibitor has been described in detail, the pseudotrisaccharide allosamidin. Recently, however, two potent natural-product cyclopentapeptide chitinase inhibitors, argifin and argadin, were reported. Here, we describe high- resoln. crystal structures that reveal the details of the interactions of these cyclopeptides with a family 18 chitinase. The structures are examples of complexes of a carbohydrate-processing enzyme with high-affinity peptide-based inhibitors and show in detail how the peptide backbone and side chains mimic the interactions of the enzyme with chitooligosaccharides. Together with enzymol. characterization, the structures explain why argadin shows an order of magnitude stronger inhibition than allosamidin, whereas argifin shows weaker inhibition. The peptides bind to the chitinase in remarkably different ways, which may explain the differences in inhibition consts. The two complexes provide a basis for structure-based design of potent chitinase inhibitors, accessible by std. peptide chem. Y1 - 2002 ER - TY - JOUR A1 - Eijsink, Vincent G. H. A1 - Synstad, Bjoenar A1 - Gaseidnes, Sigrid A1 - Komander, David A1 - Houston, Douglas R. A1 - Peter, Martin G. A1 - van Aalten, Daan M. F. T1 - Structure and function of chitinolytic enzymes N2 - The recent work on a variety of family 18 chitonolytic enzymes has yielded important data concerning the structure, substrate-binding, catalysis, inhibitor-binding and even dynamics. These data have been useful in helping to better understand the roles of various types of chitinases in chitin hydrolysis, to rationally engineer the properties of these enzymes, thus making them more suitable as biocatalysts, and to study and understand the effectiveness of natural and designed chitinase inhibitors, which may be of medical interest. On the other hand, the recent work on ChiB shows that catalysis in family 18 chitinases is a highly complicated process, involving larger parts of the enzyme and dynamics. Thus, despite recent discoveries, there is still a lot more to discover about how these enzyme work. Y1 - 2003 SN - 82-471-5901-5 ER - TY - JOUR A1 - Cederkvist, F. Henning A1 - Zamfir, Alina D. A1 - Bahrke, Sven A1 - Eijsink, Vincent G. H. A1 - Sorlie, Morten A1 - Peter-Katalinic, Jasna A1 - Peter, Martin G. T1 - Identification of a high-affinity-binding oligosaccharide by (+) nanoelectrospray quadrupole time-of-flight tandem mass spectrometry of a noncovalent enzyme-ligand complex Y1 - 2006 UR - http://www3.interscience.wiley.com/cgi-bin/jhome/26737/ U6 - https://doi.org/10.1002/anie.200503168 SN - 1433-7851 ER -