Next generation sequencing was used to identify Notch mutations in a large collection of diverse solid tumors. NOTCH1 and NOTCH2 rearrangements leading to constitutive receptor activation were confined to triple negative breast cancers (TNBC, 6 of 66 tumors). TNBC cell lines with NOTCH1 rearrangements associated with high levels of activated NOTCH1 (N1-ICD) were sensitive to the gamma-secretase inhibitor (GSI) MRK-003, both alone and in combination with pacitaxel, in vitro and in vivo, whereas cell lines with NOTCH2 rearrangements were resistant to GSI. Immunohistochemical staining of N1-ICD in TNBC xenografts correlated with responsiveness, and expression levels of the direct Notch target gene HES4 correlated with outcome in TNBC patients. Activating NOTCH1 point mutations were also identified in other solid tumors, including adenoid cystic carcinoma (ACC). Notably, ACC primary tumor xenografts with activating NOTCH1 mutations and high N1-ICD levels were sensitive to GSI, whereas N1-ICD-low tumors without NOTCH1 mutations were resistant.

Shikimate kinase, despite low sequence identity, has been shown to be structurally a member of the nucleoside monophosphate (NMP) kinase family, which includes adenylate kinase. In this paper we have explored the roles of residues in the P-loop of shikimate kinase, which forms the binding site for nucleotides and is one of the most conserved structural features in proteins. In common with many members of the P-loop family, shikimate kinase contains a cysteine residue 2 amino acids upstream of the essential lysine residue; the side chains of these residues are shown to form an ion pair. The C13S mutant of shikimate kinase was found to be enzymatically active, whereas the K15M mutant was inactive. However, the latter mutant had both increased thermostability and affinity for ATP when compared to the wild-type enzyme. The structure of the K15M mutant protein has been determined at 1.8 Å, and shows that the organization of the P-loop and flanking regions is heavily disturbed. This indicates that, besides its role in catalysis, the P-loop lysine also has an important structural role. The structure of the K15M mutant also reveals that the formation of an additional arginine/aspartate ion pair is the most likely reason for its increased thermostability. From studies of ligand binding it appears that, like adenylate kinase, shikimate kinase binds substrates randomly and in a synergistic fashion, indicating that the two enzymes have similar catalytic mechanisms.

Shikimate dehydrogenase catalyzes the fourth step of the shikimate pathway, the essential route for the biosynthesis of aromatic compounds in plants and microorganisms. Absent in metazoans, this pathway is an attractive target for nontoxic herbicides and drugs. Escherichia coli expresses two shikimate dehydrogenase paralogs, the NADP-specific AroE and a putative enzyme YdiB. Here we characterize YdiB as a dual specificity quinate/shikimate dehydrogenase that utilizes either NAD or NADP as a cofactor. Structures of AroE and YdiB with bound cofactors were determined at 1.5 and 2.5 Å resolution, respectively. Both enzymes display a similar architecture with two ␣/␤ domains separated by a wide cleft. Comparison of their dinucleotide-binding domains reveals the molecular basis for cofactor specificity. Independent molecules display conformational flexibility suggesting that a switch between open and closed conformations occurs upon substrate binding. Sequence analysis and structural comparison led us to propose the catalytic machinery and a model for 3-dehydroshikimate recognition. Furthermore, we discuss the evolutionary and metabolic implications of the presence of two shikimate dehydrogenases in E. coli and other organisms.The shikimate pathway, which links metabolism of carbohydrates to biosynthesis of aromatic compounds, is essential to plants, bacteria, and fungi (1) as well as apicomplexan parasites (2). This seven-step metabolic route leads from phosphoenolpyruvate and erythrose 4-phosphate to chorismate, the common precursor for the synthesis of folic acid, ubiquinone, vitamins E and K, and aromatic amino acids (1). This pathway is absent in metazoans, which must obtain the essential amino acids phenylalanine and tryptophan from their diet. Therefore, enzymes of this pathway are important targets for the development of nontoxic herbicides (3), as well as antimicrobial (4) and antiparasite (2) agents. The sixth step in the pathway, catalyzed by 5-enolpyruvylshikimate-3-phosphate synthase, has already been successfully targeted, with the development of glyphosate, a broad spectrum herbicide (5). However, after 20 years of extensive use, glyphosate-resistant weeds have recently emerged (6), emphasizing the importance of maintaining target diversity. In order to design new inhibitors, crystal structures of several enzymes of the shikimate pathway have been elucidated recently: 3-dehydroquinate synthase (7), type I and II dehydroquinases (8), type I and II shikimate kinases (9, 10), and 5-enolpyruvylshikimate-3-phosphate synthase (11), catalyzing the second, third, fifth, and sixth steps of the pathway, respectively.Shikimate dehydrogenase (EC 1.1.1.25) catalyzes the fourth reaction in the shikimate pathway, the NADP-dependent reduction of 3-dehydroshikimate to shikimate (Fig. 1A). Whereas dehydrogenases usually form oligomers, shikimate dehydrogenase, coded by the gene aroE in Escherichia coli, is present as a monomer in most bacteria (12, 13). In higher organisms this activity is part of a multifunctional enzym...

The crystal structure of chorismate synthase (CS) from Streptococcus pneumoniae has been solved to 2.0 A resolution in the presence of flavin mononucleotide (FMN) and the substrate 5-enolpyruvyl-3-shikimate phosphate (EPSP). CS catalyses the final step of the shikimate pathway and is a potential therapeutic target for the rational design of novel antibacterials, antifungals, antiprotozoals, and herbicides. CS is a tetramer with the monomer possessing a novel beta-alpha-beta fold. The interactions between the enzyme, cofactor, and substrate reveal the structural reasons underlying the unique catalytic mechanism and identify the amino acids involved. This structure provides the essential initial information necessary for the generation of novel anti-infective compounds by a structure-guided medicinal chemistry approach.

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