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 ؊2 to ؉3) shows closure of the ''roof'' of the active site tunnel. It also shows that the sugar in the ؊1 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 ؉1 and ؉2 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.C hitinases hydrolyze chitin, a linear polymer of ␤-(1,4)-linked N-acetylglucosamine (NAG), which is an abundant biopolymer. These enzymes are essential to chitin-containing organisms (fungi, insects, crustaceans) and are used by several bacteria to exploit chitin as a source of energy. Chitinase inhibitors have generated a lot of interest given their potential as insecticides (1), fungicides (2, 3), and antimalarials (4, 5). Biotechnological exploitation of chitinases, as well as design of inhibitors with sufficiently high selectivity and affinity, requires detailed knowledge of the catalytic mechanism and enzyme-substrate interactions.Most nonplant chitinases belong to glycosidase family 18 (6) and degrade chitin with retention of the stereochemistry at the anomeric carbon (7-10). The reaction is thought to be initiated by distortion of the Ϫ1 sugar ring and protonation of the glycosidic oxygen by a protonated acidic residue. The subsequent nucleophilic attack differs from classical reaction mechanisms of retaining enzymes such as lysozyme (11) and amylases (12) in that it involves the N-acetyl group of the Ϫ1 sugar, rather than a carboxylate side chain on the protein (8,9,13,14). Thus, the first step of chitinolysis results in cleavage of the sugar chain and formation of an oxazolinium ion intermediate, and hydrolysis of this ion completes the reaction (9, 15) (Fig. 1).Although the current model for the catalytic mechanism is well established, the amount of structural evidence in its support is limited (8). Important elements of the proposed mechanism were inferred from modeling studies and structures of glycosidases that do not belong to f...

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The p16/RB/E2F regulatory pathway, which controls transit through the G1 restriction point of the cell cycle, is one of the most frequent targets of genetic alterations in human cancer. Any of these alterations results in the deregulated expression of the transcription factor E2F, one of the key mediators of cell cycle progression. Under these conditions, E2F1 also participates in the induction of apoptosis by a p53-dependent pathway, and independently of p53. Recently, we identified the p53-homolog p73 as a first direct target of p53-independent apoptosis. Here, we used a cDNA microarray to screen an inducible E2F1-expressing Saos-2 cell line for E2F1 target genes. Expression analysis by cDNA microarray and RT-PCR revealed novel E2F1 target genes involved in E2F1-regulated cellular functions such as cell cycle control, DNA replication and apoptosis. In addition, the identification of novel E2F1 target genes participating in the processes of angiogenesis, invasion and metastasis supports the view that E2F1 plays a central role in many aspects of cancer development. These results provide new insight into the role of E2F1 in tumorigenesis as a basis for the development of novel anti-cancer therapeutics.

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Many quinones and their precursors, which are transformed oxidatively into quinones and/or quinone methides, are important natural products. As secondary metabolites, they frequently possess antibiotic and cytotoxic activities, in addition to acting sometimes as pathogens. Several plants and animals, especially insects, use quinonoid substances for defense, often with spectacular results. On the macromolecular level, quinone methides have a key role in the plant kingdom in lignin biosynthesis; the biosynthesis of melanoproteins exemplifies the reactions of o-quinones in the animal kingdom. In insects, cross-linking of structural proteins through quinones and quinone methides results in the construction of the sclerotized exoskeleton. For mankind, the reactivity of quinones in biological systems has far-reaching consequences of pharmaceutical, toxicological, and technical relevance. The examples in this review show that a common principle underlies these reactions, namely, the chemical modification of biopolymers. As demonstrated particularly well in a more detailed discussion of the chemical principles of insect cuticle sclerotization, several major and important new results have emerged from the development and applications of modern methods of sample separation and from solid-state NMR spectroscopy.