DAHP synthase and chorismate mutase catalyze key steps in the shikimate biosynthetic pathway en route to aromatic amino acids. In Mycobacterium tuberculosis, chorismate mutase (MtCM; Rv0948c), located at the branch point toward phenylalanine and tyrosine, has poor activity on its own. However, it is efficiently activated by the first enzyme of the pathway, DAHP synthase (MtDS; Rv2178c), through formation of a non-covalent MtCM–MtDS complex. Here, we show how MtDS serves as an allosteric platform for feedback regulation of both enzymes, using X-ray crystallography, small-angle X-ray scattering, size-exclusion chromatography, and multi-angle light scattering. Crystal structures of the fully inhibited MtDS and the allosterically down-regulated MtCM–MtDS complex, solved at 2.8 and 2.7Å, respectively, reveal how effector binding at the internal MtDS subunit interfaces regulates the activity of MtDS and MtCM. While binding of all three metabolic end products to MtDS shuts down the entire pathway, the binding of phenylalanine jointly with tyrosine releases MtCM from the MtCM–MtDS complex, hence suppressing MtCM activation by 'inter-enzyme allostery'. This elegant regulatory principle, invoking a transient allosteric enzyme interaction, seems to be driven by dynamics and is likely a general strategy used by nature.

Papain-like cysteine proteases (PLCPs) constitute the largest group of thiol-based protein degrading enzymes and are characterized by a highly conserved fold. They are found in bacteria, viruses, plants and animals and involved in a number of physiological and pathological processes, parasitic infections and host defense, making them interesting targets for drug design. The Marasmius oreades agglutinin (MOA) is a blood group B-specific fungal chimerolectin with calcium-dependent proteolytic activity. The proteolytic domain of MOA presents a unique structural arrangement, yet mimicking the main structural elements in known PLCPs. Here we present the X-ray crystal structure of MOA in complex with Z-VAD-fmk, an irreversible caspase inhibitor known to cross-react with PLCPs. The structural data allow modeling of the substrate binding geometry and mapping of the fundamental enzyme-substrate interactions. The new information consolidates MOA as a new, yet strongly atypical member of the papain superfamily. The reported complex is the first published structure of a PLCP in complex with the well characterized caspase inhibitor Z-VAD-fmk.

The crystal structure of the α-galactosyl binding Lyophyllum decastes lectin (LDL) was determined to 1.0 Å resolution by sulfur single-wavelength anomalous diffraction (SAD). The 10 kDa protein exhibits no sequence similarity to any protein with known structure and adopts a unique lectin fold, where a core of two antiparallel β-sheets at the heart of the homodimer is connected to the periphery of the structure by intramolecular disulfide bridges. This fold suggests that LDL is secreted, which sets it apart from other mushroom lectins. Structures of complexes between LDL and the ligands α-methylgalactoside and globotriose shed light on the binding specificity. Sequence comparison suggests a location and function of LDL and homologous proteins in or at the fungal cell wall. Structural comparison allows the identification of a superfamily of secreted proteins with the LDL fold, which may play a role at the interface between fungi and their environment.

For more than half a century, transition state theory has provided a useful framework for understanding the origins of enzyme catalysis. As proposed by Pauling, enzymes accelerate chemical reactions by binding transition states tighter than substrates, thereby lowering the activation energy compared with that of the corresponding uncatalyzed process. This paradigm has been challenged for chorismate mutase (CM), a well-characterized metabolic enzyme that catalyzes the rearrangement of chorismate to prephenate. Calculations have predicted the decisive factor in CM catalysis to be ground state destabilization rather than transition state stabilization. Using X-ray crystallography, we show, in contrast, that a sluggish variant of Bacillus subtilis CM, in which a cationic active-site arginine was replaced by a neutral citrulline, is a poor catalyst even though it effectively preorganizes chorismate for the reaction. A series of high-resolution molecular snapshots of the reaction coordinate, including the apo enzyme, and complexes with substrate, transition state analog and product, demonstrate that an active site, which is only complementary in shape to a reactive substrate conformer, is insufficient for effective catalysis. Instead, as with other enzymes, electrostatic stabilization of the CM transition state appears to be crucial for achieving high reaction rates.

Tuberculosis (TB) is a devastating disease that claims millions of lives every year. Hindered access or non-compliance to medication, especially in developing countries, led to drug resistance, further aggravating the situation. With current standard therapies in use for over 50 years and only few new candidates in clinical trials, there is an urgent call for new TB drugs. A powerful tool for the development of new medication is structure-guided design, combined with virtual screening or docking studies. Here, we report the results of a drug-design project, which we based on a publication that claimed the structure-guided discovery of several promising and highly active inhibitors targeting the secreted chorismate mutase (*MtCM) from Mycobacterium tuberculosis. We set out to further improve on these compounds and synthesized a series of new derivatives. Thorough evaluation of these molecules in enzymatic assays revealed, to our dismay, that neither the claimed lead compounds, nor any of the synthesized derivatives, show any inhibitory effects against *MtCM.

A reliable, oral cure for hepatitis C infection is still a major unmet medical need.The approval of the NS3 protease inhibitors, telaprevir and boceprevir, in 2011 has been a welcome step in the right direction, but removing the need for pegylated interferon and ribavirin to suppress the emergence of resistance mutations remains an issue.Future treatment is expected to require a combination of drugs acting via different mechanisms.By applying our proprietary fragment screening platform, Pyramid™, to the crystal structure of the full length NS3/4a protein we identified a novel allosteric site at the protease-helicase interface, distinct from the active site targeted by the recently launched drugs boceprevir and telaprevir.Using structure-based design strategies, the initial weakly binding fragment hits were optimised against the full length NS3/4a protein, resulting in leads with low nM potency.Potent compounds demonstrated robust anti-viral activity when evaluated in the cell-based genotype 1b sub-genomic replicon assay, with a wide cytotoxicity window.The physicochemical properties of these early leads were then optimised to gain suitable pharmacokinetics for oral dosing.The resulting key compounds with sub 10nM potency in the replicon assay have MW <400 and cLogP <2a distinct contrast to many known protease inhibitors.During this conference we will show the allosteric binding site with bound compounds and the rationale for targeting this site.Key replicon data, including combination studies, will be shown along with pharmacokinetic data for our preclinical compounds.

Aldolases are enzymes with potential applications in biosynthesis, depending on their activity, specificity and stability. In the present study, the genomes of Sulfolobus species were screened for aldolases. Two new KDGA [2-keto-3-deoxygluconate (2-oxo-3-deoxygluconate) aldolases] from Sulfolobus acidocaldarius and Sulfolobus tokodaii were identified, overexpressed in Escherichia coli and characterized. Both enzymes were found to have biochemical properties similar to the previously characterized S. solfataricus KDGA, including the condensation of pyruvate and either D,L-glyceraldehyde or D,L-glyceraldehyde 3-phosphate. The crystal structure of S. acidocaldarius KDGA revealed the presence of a novel phosphate-binding motif that allows the formation of multiple hydrogen-bonding interactions with the acceptor substrate, and enables high activity with glyceraldehyde 3-phosphate. Activity analyses with unnatural substrates revealed that these three KDGAs readily accept aldehydes with two to four carbon atoms, and that even aldoses with five carbon atoms are accepted to some extent. Water-mediated interactions permit binding of substrates in multiple conformations in the spacious hydrophilic binding site, and correlate with the observed broad substrate specificity.

Invasion of epithelial cells by Shigella flexneri is characterized by cytoskeletal rearrangements of the host cell membrane, promoting internalization of the bacterium. The bacterial effector IpaA is injected into the epithelial cell by a type III secretion apparatus and recruits vinculin to regulate actin polymerization at the site of entry. We analysed the complex formed between a carboxy‐terminal fragment of IpaA (IpaA 560−633 ) and the vinculin D1 domain (VD1), both in crystals and in solution. We present evidence that IpaA 560−633 has two α‐helical vinculin‐binding sites that simultaneously bind two VD1 molecules. The interaction of IpaA 560−633 with VD1 is highly similar to the interaction of the endogenous, eukaryotic proteins talin and α‐actinin with VD1, showing that Shigella uses a structural mimicry strategy to activate vinculin.

Escherichia coli bacterioferritin was serendipitously crystallized in a novel cubic crystal form and its structure could be determined to 2.5 angstrom resolution despite a high degree of merohedral twinning. This is the first report of crystallographic data on `as-isolated' E. coli bacterioferritin. The ferroxidase active site contains positive difference density consistent with two metal ions that had co-purified with the protein. X-ray fluorescence studies suggest that the metal composition is different from that of previous structures and is a mix of zinc and native iron ions. The ferroxidase-centre configuration displays a similar flexibility as previously noted for other bacterioferritins.