Crystal structure of mammalian selenocysteine-dependent iodothyronine deiodinase suggests a peroxiredoxin-like catalytic mechanism.

Local levels of active thyroid hormone (3,3',5-triiodothyronine) are controlled by the action of activating and inactivating iodothyronine deiodinase enzymes. Deiodinases are selenocysteine-dependent membrane proteins catalyzing the reductive elimination of iodide from iodothyronines through a poorly understood mechanism. We solved the crystal structure of the catalytic domain of mouse deiodinase 3 (Dio3), which reveals a close structural similarity to atypical 2-Cys peroxiredoxin(s) (Prx). The structure suggests a route for proton transfer to the substrate during deiodination and a Prx-related mechanism for subsequent recycling of the transiently oxidized enzyme. The proposed mechanism is supported by biochemical experiments and is consistent with the effects of mutations of conserved amino acids on Dio3 activity. Thioredoxin and glutaredoxin reduce the oxidized Dio3 at physiological concentrations, and dimerization appears to activate the enzyme by displacing an autoinhibitory loop from the iodothyronine binding site. Deiodinases apparently evolved from the ubiquitous Prx scaffold, and their structure and catalytic mechanism reconcile a plethora of partly conflicting data reported for these enzymes.

A third metal is required for catalytic activity of the signal-transducing protein phosphatase M tPphA.

Protein phosphatase M (PPM) regulates key signaling pathways in prokaryotes and eukaryotes. Novel structures of bacterial PPM members revealed three divalent metal ions in their catalytic centers. The function of metal 3 (M3) remained unclear. To reveal its function, we created variants of tPphA from Thermosynechococcus elongatus in all metal-coordinating residues, and multiple variants were created for the M3 coordinating Asp-119 residue. The structures of variants D119A and D193A were resolved, showing loss of M3 binding but unaffected binding of M1 and M2 in the catalytic center of D119A, with the nucleophilic water molecule in the correct place. The catalytic activity of this variant was highly impaired. This and further structure-function analyses showed that M3 is required for catalysis by providing a water molecule as a proton donor during catalysis. Mutation of the homologue Asp residue in human PP2Cα also caused loss of function, suggesting a general requirement of M3 in PPM-catalyzed reactions.

Expression, purification, crystallization and preliminary X-ray analysis of the DNA-binding domain of Rhodobacter capsulatus MopB.

The LysR-type regulator MopB represses transcription of several target genes (including the nitrogen-fixation gene anfA) in Rhodobacter capsulatus at high molybdenum concentrations. In this study, the isolated DNA-binding domain of MopB (MopBHTH) was overexpressed in Escherichia coli. Purified MopBHTH bound the anfA promoter as shown by DNA mobility-shift assays, demonstrating the function of the isolated regulator domain. MopBHTH was crystallized using the sitting-drop vapour-diffusion method in the presence of 0.2 M lithium sulfate, 0.1 M phosphate/citrate pH 4.2, 20%(w/v) PEG 1000 at 291 K. The crystal belonged to space group P3(1)21 or P3(2)21, with unit-cell parameters a=b=61.84, c=139.64 Å, α=ß=90, γ=120°, and diffracted to 3.3 Šresolution at a synchrotron source.

Engineering and application of a biosensor with focused ligand specificity.

Cell factories converting bio-based precursors to chemicals present an attractive avenue to a sustainable economy, yet screening of genetically diverse strain libraries to identify the best-performing whole-cell biocatalysts is a low-throughput endeavor. For this reason, transcriptional biosensors attract attention as they allow the screening of vast libraries when used in combination with fluorescence-activated cell sorting (FACS). However, broad ligand specificity of transcriptional regulators (TRs) often prohibits the development of such ultra-high-throughput screens. Here, we solve the structure of the TR LysG of Corynebacterium glutamicum, which detects all three basic amino acids. Based on this information, we follow a semi-rational engineering approach using a FACS-based screening/counterscreening strategy to generate an L-lysine insensitive LysG-based biosensor. This biosensor can be used to isolate L-histidine-producing strains by FACS, showing that TR engineering towards a more focused ligand spectrum can expand the scope of application of such metabolite sensors.

Carbonic anhydrase inhibitors. Inhibition of the beta-class enzymes from the fungal pathogens Candida albicans and Cryptococcus neoformans with aliphatic and aromatic carboxylates.

The inhibition of the beta-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic fungi Cryptococcus neoformans (Can2) and Candida albicans (Nce103) with carboxylates such as the C1-C5 aliphatic carboxylates, oxalate, malonate, maleate, malate, pyruvate, lactate, citrate and some benzoates has been investigated. The best Can2 inhibitors were acetate and maleate (K(I)s of 7.3-8.7 microM), whereas formate, acetate, valerate, oxalate, maleate, citrate and 2,3,5,6-tetrafluorobenzoate showed less effective inhibition, with K(I)s in the range of 42.8-88.6 microM. Propionate, butyrate, malonate, L-malate, pyruvate, L-lactate and benzoate, were weak Can2 inhibitors, with inhibition constants in the range of 225-1267 microM. Nce103 was more susceptible to inhibition with carboxylates compared to Can2, with the best inhibitors (maleate, benzoate, butyrate and malonate) showing K(I)s in the range of 8.6-26.9 microM. L-Malate and pyruvate together with valerate were the less efficient Nce103 inhibitors (K(I)s of 87.7-94.0 microM), while the remaining carboxylates showed a compact behavior of efficient inhibitors (K(I)s in the range of 35.1-61.6 microM). Notably the inhibition profiles of the two fungal beta-CAs was very different from that of the ubiquitous host enzyme hCA II (belonging to the alpha-CA family), with maleate showing selectivity ratios of 113.6 and 115 for Can2 and Nce103, respectively, over hCA II inhibition. Therefore, maleate is a promising starting lead molecule for the development of better, low nanomolar, selective beta-CA inhibitors.

Carbonic anhydrase inhibitors. Inhibition and homology modeling studies of the fungal beta-carbonic anhydrase from Candida albicans with sulfonamides.

The beta-carbonic anhydrase (CA, EC 4.2.1.1) from the fungal pathogen Candida albicans (Nce103) is involved in a CO(2) sensing pathway critical for the pathogen life cycle and amenable to drug design studies. Herein we report an inhibition study of Nce103 with a library of sulfonamides and one sulfamate, showing that Nce103, similarly to the related enzyme from Cryptococcus neoformans Can2, is inhibited by these compounds with K(I)s in the range of 132 nM-7.6 microM. The best Nce103 inhibitors were acetazolamide, methazolamide, bromosulfanilamide, and 4-hydroxymethylbenzenesulfonamide (K(I)s<500 nM). A homology model was generated for Nce103 based on the crystal structure of Can2. The model shows that compounds with zinc-binding groups incorporating less polar moieties and compact scaffolds generate stronger Nce103 inhibitors, whereas highly polar zinc-binding groups and bulkier compounds appear more promising for the specific inhibition of Can2. Such compounds may be useful for the design of antifungal agents possessing a new mechanism of action.

Comparison of classical serotyping and PremiTest assay for routine identification of common Salmonella enterica serovars.

The commercial PremiTest Salmonella kit uses a multiplexed DNA typing test aimed at identifying common serovars of Salmonella enterica. It was used in assays over a 9-month period in the Belgian reference laboratory that performs the routine identification of Salmonella strains of animal origin. A blind analysis of 754 strains was conducted in parallel by classical serotyping and the PremiTest assay. Full results were available for 685 strains (90.8%) by serotyping, while the remaining 69 strains were found to be nontypeable due to either a lack of surface antigen expression or autoagglutination properties. When the PremiTest assay (version 4.2) was performed with crude bacterial extracts, it identified 658 strains (87.3%), including most strains found to be nontypeable by serotyping. In contrast, it gave no, wrong, dual, or noninterpretable results for 96 strains, for which 23 were caused by assay failures. When purified DNA instead of crude extracts were tested, the number of strains successfully identified to the serovar level increased to 714 (94.7%), while all assay failures were cleared. Our conclusion is that, in its actual development stage, the application of the investigated kit to purified DNA samples offers a valuable alternative to classical serotyping for laboratories performing the routine identification of Salmonella strains belonging to commonly encountered serovars and isolated from a given geographical area, assuming that the system has been validated beforehand with a significant number of strains originating from that particular area.

A molecular mechanism for direct sirtuin activation by resveratrol.

Sirtuins are protein deacetylases regulating metabolism, stress responses, and aging processes, and they were suggested to mediate the lifespan extending effect of a low calorie diet. Sirtuin activation by the polyphenol resveratrol can mimic such lifespan extending effects and alleviate metabolic diseases. The mechanism of Sirtuin stimulation is unknown, hindering the development of improved activators. Here we show that resveratrol inhibits human Sirt3 and stimulates Sirt5, in addition to Sirt1, against fluorophore-labeled peptide substrates but also against peptides and proteins lacking the non-physiological fluorophore modification. We further present crystal structures of Sirt3 and Sirt5 in complex with fluorogenic substrate peptide and modulator. The compound acts as a top cover, closing the Sirtuin's polypeptide binding pocket and influencing details of peptide binding by directly interacting with this substrate. Our results provide a mechanism for the direct activation of Sirtuins by small molecules and suggest that activators have to be tailored to a specific Sirtuin/substrate pair.

Structure-based development of novel sirtuin inhibitors.

Sirtuins are NAD+-dependent protein deacetylases regulating metabolism, stress responses, and aging processes. Mammalia possess seven Sirtuin isoforms, Sirt1-7, which differ in their subcellular localization and in the substrate proteins they deacetylate. The physiological roles of Sirtuins and their potential use as therapeutic targets for metabolic and aging-related diseases have spurred interest in the development of small-molecule Sirtuin modulators. Here, we describe an approach exploiting the structures available for four human Sirtuins for the development of isoform-specific inhibitors. Virtual docking of a compound library into the peptide binding pockets of crystal structures of Sirt2, 3, 5 and 6 yielded compounds potentially discriminating between these isoforms. Further characterization in activity assays revealed several inhibitory compounds with little isoform specificity, but also two compounds with micromolar potency and high specificity for Sirt2. Structure comparison and the predicted, shared binding mode of the Sirt2-specific compounds indicate a pocket extending from the peptide-binding groove as target side enabling isoform specificity. Our family-wide structure-based approach thus identified potent, Sirt2-specific inhibitors as well as lead structures and a target site for the development of compounds specific for other Sirtuin isoform, constituting an important step toward the identification of a complete panel of isoform-specific Sirtuin inhibitors.

Structure and inhibition of the CO2-sensing carbonic anhydrase Can2 from the pathogenic fungus Cryptococcus neoformans.

In the pathogenic fungus Cryptococcus neoformans, a CO(2)-sensing system is essential for survival in the natural environment (approximately 0.03% CO(2)) and mediates the switch to virulent growth in the human host (approximately 5% CO(2)). This system is composed of the carbonic anhydrase (CA) Can2, which catalyzes formation of bicarbonate, and the fungal, bicarbonate-stimulated adenylyl cyclase Cac1. The critical role of these enzymes for fungal metabolism and pathogenesis identifies them as targets for antifungal drugs. Here, we prove functional similarity of Can2 to the CA Nce103 from Candida albicans and describe its biochemical and structural characterization. The crystal structure of Can2 reveals that the enzyme belongs to the "plant-type" beta-CAs but carries a unique N-terminal extension that can interact with the active-site entrance of the dimer. We further tested a panel of compounds, identifying nanomolar Can2 inhibitors, and present the structure of a Can2 complex with the inhibitor and product analog acetate, revealing insights into interactions with physiological ligands and inhibitors.

Substrates and regulation mechanisms for the human mitochondrial sirtuins Sirt3 and Sirt5.

The enzymes of the Sirtuin family of nicotinamide-adenine-dinucleotide-dependent protein deacetylases are emerging key players in nuclear and cytosolic signaling, but also in mitochondrial regulation and aging. Mammalian mitochondria contain three Sirtuins, Sirt3, Sirt4, and Sirt5. Only one substrate is known for Sirt3 as well as for Sirt4, and up to now, no target for Sirt5 has been reported. Here, we describe the identification of novel substrates for the human mitochondrial Sirtuin isoforms Sirt3 and Sirt5. We show that Sirt3 can deacetylate and thereby activate a central metabolic regulator in the mitochondrial matrix, glutamate dehydrogenase. Furthermore, Sirt3 deacetylates and activates isocitrate dehydrogenase 2, an enzyme that promotes regeneration of antioxidants and catalyzes a key regulation point of the citric acid cycle. Sirt3 thus can regulate flux and anapleurosis of this central metabolic cycle. We further find that the N- and C-terminal regions of Sirt3 regulate its activity against glutamate dehydrogenase and a peptide substrate, indicating roles for these regions in substrate recognition and Sirtuin regulation. Sirt5, in contrast to Sirt3, deacetylates none of the mitochondrial matrix proteins tested. Instead, it can deacetylate cytochrome c, a protein of the mitochondrial intermembrane space with a central function in oxidative metabolism, as well as apoptosis initiation. Using a mitochondrial import assay, we find that Sirt5 can indeed be translocated into the mitochondrial intermembrane space, but also into the matrix, indicating that localization might contribute to Sirt5 regulation and substrate selection.

Structural analysis of the PP2C phosphatase tPphA from Thermosynechococcus elongatus: a flexible flap subdomain controls access to the catalytic site.

The homologue of the phosphoprotein PII phosphatase PphA from Thermosynechococcus elongatus, termed tPphA, was identified and its structure was resolved in two different space groups, C222(1) and P4(1)2(1)2, at a resolution of 1.28 and 3.05 A, respectively. tPphA belongs to a large and widely distributed subfamily of Mg(2+)/Mn(2+)-dependent phosphatases of the PPM superfamily characterized by the lack of catalytic and regulatory domains. The core structure of tPphA shows a high degree of similarity to the two PPM structures identified so far. In contrast to human PP2C, but similar to Mycobacterium tuberculosis phosphatase PstP, the catalytic centre exhibits a third metal ion in addition to the dinuclear metal centre universally conserved in all PPM members. The fact that the third metal is only liganded by amino acids, which are universally conserved in all PPM members, implies that the third metal could be general for all members of this family. As a specific feature of tPphA, a flexible subdomain, previously recognized as a flap domain, could be revealed. Comparison of different structural isomers of tPphA as well as site-specific mutagenesis implied that the flap domain is involved in substrate binding and catalytic activity. The structural arrangement of the flap domain was accompanied by a large side-chain movement of an Arg residue (Arg169) at the basis of the flap. Mutation of this residue strongly impaired protein stability as well as catalytic activity, emphasizing the importance of this amino acid for the regional polysterism of the flap subdomain and confirming the assumption that flap domain flexibility is involved in catalysis.

Structure-based development of novel adenylyl cyclase inhibitors.

In mammals, the second messenger cAMP is synthesized by a family of transmembrane isoforms (tmACs) and one known cytoplasmic enzyme, "soluble" adenylyl cyclase (sAC). Understanding the individual contributions of these families to cAMP signaling requires tools which can distinguish them. Here, we describe the structure-based development of isoform discriminating AC inhibitors. Docking calculations using a library of small molecules with the crystal structure of a sAC homologue complexed with the noncompetitive inhibitor catechol estrogen identified two novel inhibitors, 3,20-dioxopregn-4-en-21-yl4-bromobenzenesulfonate (2) and 1,2,3,4,5,6,7,8,13,13,14,14-dodecachloro-1,4,4a,4b,5,8,8a,12b-octahydro-11-sulfo-1,4:5,8-dimethanotriphenylene-10-carboxylic acid (3). In vitro testing revealed that 3 defines a novel AC inhibitor scaffold with high affinity for human sAC and less inhibitory effect on mammalian tmACs. 2 also discriminates between sAC and tmACs, and it appears to simultaneously block the original binding pocket and a neighboring interaction site. Our results show that compounds exploiting the catechol estrogen binding site can produce potent, isoform discriminating AC inhibitors.

The C2A-C2B linker defines the high affinity Ca(2+) binding mode of rabphilin-3A.

The Ca(2+) binding properties of C2 domains are essential for the function of their host proteins. We present here the first crystal structures showing an unexpected Ca(2+) binding mode of the C2B domain of rabphilin-3A in atomic detail. Acidic residues from the linker region between the C2A and C2B domains of rabphilin-3A interact with the Ca(2+)-binding region of the C2B domain. Because of these interactions, the coordination sphere of the two bound Ca(2+) ions is almost complete. Mutation of these acidic residues to alanine resulted in a 10-fold decrease in the intrinsic Ca(2+) binding affinity of the C2B domain. Using NMR spectroscopy, we show that this interaction occurred only in the Ca(2+)-bound state of the C2B domain. In addition, this Ca(2+) binding mode was maintained in the C2 domain tandem fragment. In NMR-based liposome binding assays, the linker was not released upon phospholipid binding. Therefore, this unprecedented Ca(2+) binding mode not only shows how a C2 domain increases its intrinsic Ca(2+) affinity, but also provides the structural base for an atypical protein-Ca(2+)-phospholipid binding mode of rabphilin-3A.