An Application of Declarative Languages in Distributed Architectures: ASP and DALI Microservices

In this paper we introduce an approach to the possible adoption of Answer Set Programming (ASP) for the definition of microservices, which are a successful abstraction for designing distributed applications as suites of independently deployable interacting components. Such ASP-based components might be employed in distributed architectures related to Cloud Computing or to the Internet of Things (IoT), where the ASP microservices might be usefully coordinated with intelligent logic-based agents. We develop a case study where we consider ASP microservices in synergy with agents defined in DALI, a well-known logic-based agent-oriented programming language developed by our research group

Structural And Functional Characterization Of Proteins Of Unknown Function (hp0495, Hp0100 And Hp1259) In Helicobacter Pylori

H. pylori is missing the glutaminyl- and asparaginyl-tRNA synthetases (GlnRS and AsnRS, respectively). Consequently, H. pylori uses an indirect aminoacylation pathway to generate Gln-tRNAGln and Asn-tRNAAsn. Within this process, Asn-tRNAAsn is produced by misacylation of tRNAAsn with aspartate by a non-discriminating aspartyl-tRNA synthetase (ND-AspRS). Next, the heterotrimeric, glutamine-dependent amidotransferase (called AdT or GatCAB) converts the misacylated Asp-tRNAAsn into Asn-tRNAAsn. A parallel pathway exists for the synthesis of Gln-tRNAGln, wherein misacylation of tRNAGln with glutamate is catalyzed by a tRNAGln-specific glutamyl-tRNA synthetase (GluRS2) to generate Glu-tRNAGln; this misacylated intermediate is converted to Gln-tRNAGln by the same AdT. This dependence on misacylated intermediates for protein synthesis suggests a requirement for additional mechanisms to prevent the misincorporation of Glu and Asp into proteins in place of Gln and Asn. H. pylori elongation factor (EF-Tu) provides one such machinery, but it is not sufficient to maintain translational accuracy, suggesting the need for additional mechanisms. Hp0495 and Hp0100 were identified by yeast two-hybrid (Y2H) as potential new players in tRNA aminoacylation and fidelity. By Y2H, Hp0495 showed interactions with EF-Tu and Hp0100 was connected to both ND-AspRS and AdT. The work presented in this dissertation examined the possible roles of both of these proteins in indirect aminoacylation and in promoting tRNA accuracy. Using SPR, native gels, and size exclusion chromatography, we have shown that Hp0495 forms complexes with EF-Tu in both its GDP and GTP forms, but preferentially binds EF-Tu*GTP. In collaboration with a colleague, Dr. Keng-Ming Chang, we have discovered that Hp0495 binds ATP, glutamate, and either tRNAGlu1 or tRNAGln and forms tRNAGln-dependent complex with GluRS2. Also, Hp0495 binds deacylated tRNAs more tightly than aminoacylated tRNAs for the five cases tested in this work. Hp0495 has similarities to ACT/RAM domains; these domains typically regulate different aspects of amino acid and nucleotide metabolism. Our hypothesis is that Hp0495 might be a regulatory protein, sensing tRNAGlu1/tRNAGln and directing one or both it to either GluRS1/GluRS2 for aminoacylation or for functions outside protein translation. The hp0495 gene is in an operon with mraY and murD, these two enzymes are involved in cell wall biosynthesis, suggesting that Hp0495 might be involved in regulating this process. There is also precedence for the involvement of EF-Tu in cell wall biosynthesis, supporting this hypothesis. In collaboration with a colleague, Dr. Gayathri Silva, we have demonstrated that Hp0100 is, in fact, an essential component of a tRNA-independent Asn-transamidosome complex. This complex contains ND-AspRS, AdT, and Hp0100 and facilitates AdT\u27s transamidation process of Asp-tRNAAsn (~ 35-fold) and Glu-tRNAGln (~ 3-fold). Our work characterizing the Asn-transamidosome and transamidation of Asp-tRNAAsn was published in 2013 (Silva, G. N., Fatma, S., et al. J. Biol. Chem. 288, 3816). An hp0100 knockout suggests that Hp0100 is not essential under robust growth conditions. Our revised hypothesis is that the growth conditions used in these experiments were too permissive and did not adequately reflect the stress conditions faced by H. pylori in its human niche. Viability of our hp0100 strain will be tested under different stress conditions with the goal of identifying conditions where Hp0100 is conditionally essential. Finally, we have initiated the characterization of third protein of unknown function, Hp1259. Preliminary data suggest that it might be involved in the indirect aminoacylation of tRNAGln. In the H. pylori genome, 499 open reading frames (ORFs) are annotated as hypothetical proteins whose functions are not known, including Hp0100 and Hp0495. We have assigned an important function to Hp0100, namely promoting tRNA aminoacylation, and have identified a new functional characteristics for Hp0495 that clearly demonstrate its role in tRNA aminoacylation and/or function. The existence of these proteins of unknown function and their interactions with components of the translation machinery clearly demonstrate that the classical boundaries of the field of tRNA aminoacylation need to be expanded

The cystobactamide pathway : enzymological investigation of unusual non-ribosomal peptide biosynthesis mechanisms

This thesis focused on the investigation of unusual biosynthetic reactions in the Cystobactamide pathway. Cystobactamides present an unusual hexapeptidic backbone mostly constituted from modified para-amino benzoic building blocks and a unique methoxy-isoasparaginyl moiety. Furthermore, the extensive in trans tailoring makes this pathway particularly interesting from an enzymological point of view. The present work details the in trans biosynthesis of the isopropoxyl decorations carried out on the two last para-amino benzoate units through in vitro reconstitution of these biochemical reactions including the generation of fully functional non-ribosomal peptide synthetase modules in vitro. Furthermore an in-depth biochemical investigation of the unprecedented bifunctional non-ribosomal peptide synthetase domain leading to the isomerization or dehydration of asparagine was performed in parallel with the characterization of the in trans hydroxylation and of the shuttling of this moiety. The self-resistance mechanisms of the producer strain Cystobacter velatus 34 were also investigated in comparison to the self-resistance mechanism for the related antibiotic Albicidin. Finally, attempts at complete in vitro reconstitution of the pathways were performed using the unique heterologous expression platform for non-ribosomal peptide synthetase modules developed during this thesis.Diese Dissertation beschäftigt sich mit der Untersuchung ungewöhnlicher biosynthetischer Reaktionen im Cystobactamid-Biosyntheseweg. Cystobactamide haben eine hexapeptidische Grundstruktur, die hauptsächlich aus modifizierten para-Aminobenzoesäure Bausteinen und einer einzigartigen Methoxyisoasparaginyleinheit besteht. Außerdem macht die extensive Verwendung von in trans enzymatischen Modifikationen diesen Biosyntheseweg aus enzymologischer Sichtweise besonders interessant. Die vorliegende Arbeit untersucht durch in vitro Rekonstitution dieser biochemischen Reaktionen, die in trans Biosynthese der Isopropoxyl Dekorationen, die auf den beiden letzten para-Aminobenzoateinheiten entsteht, einschließlich der Erzeugung voll funktionsfähiger nicht ribosomaler Peptidsynthetase-Module in vitro. Außerdem wurde eine umfassende biochemische Analyse der neuen bifunktionellen nicht ribosomalen Peptidsynthetase Domäne durchgeführt, die zur Isomerisierung oder Dehydratisierung von Asparagin führte, parallel zur Charakterisierung der in trans Hydroxylierung und des Transfer dieses Moleküls. Die Eigenresistenzmechanismen des Produzenten Cystobacter velatus 34 wurden im Vergleich zum Selbstresistenzmechanismus des verwandten Antibiotikums Albicidin untersucht. Letztendlich wurden Versuche einer kompletten in vitro Rekonstitution des Biosyntheseweges durchgeführt, mit Hilfe der in dieser Dissertation neuentwickelten heterologen Expressionsplattform für nicht ribosomale Peptidsynthetase-Module

Study of a Bacillus circulans chitin-binding domain by a green fluorescent protein binding assay and detection of lysozymes by improved zymograms

A fluorescent binding assay was developed to investigate the effects of site-directed mutagenesis on the binding affinity and binding specificity of the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12. The chitin-binding domain (ChBD) was genetically fused to the N-terminus of the green fluorescent protein, GFP. The polyhistidine-tagged hybrid protein was expressed in Escherichia coli under the dose-dependent regulation of the araBAD promoter and purified using metal affinity-, chitin- or ion-exchange chromatography. Residues suggested to be involved in binding from previous three-dimensional studies were mutated and their contributions to binding and substrate specificity were evaluated by depletion assays. Purified fusion proteins were incubated with chitin beads, polysaccharide-protein complexes were removed by centrifugation and the free protein concentration was measured fluorometrically. The experimental binding isotherms were analyzed by non-linear regression using a modified Langmuir equation. Binding affinity and specificity were alternatively studied by affinity electrophoresis under non-denaturing conditions. Non-conservative substitution of tryptophan residue (W687) with alanine abolished chitin-binding affinity. Double mutation E668K/P689A also impaired binding significantly. Other substitutions in the binding site had little effect on overall affinity for chitin. Interestingly, mutation T682A led to a higher specificity towards chitinous substrates than observed for the wild-type. Furthermore, the ChBD-GFP hybrid protein proved to be useful for specifically labeling cell walls of fungi and yeast and for the detection of fungal infections in tissue samples. Additionally, an improved method for detecting cell lytic activity by a colorbased zymogram was developed. Proteins were separated by electrophoresis in SDS-polyacrylamide gels copolymerized with Remazol-brilliant-blue labeled whole cells of Micrococcus lysodeikticus. After electrophoresis, the enzymes were allowed to refold and lyse the blue-labeled cells embedded in the gel, producing clearing zones in an otherwise bluish gel. This improved zymogram method allows the rapid, sensitive and simultaneous determination of cell lytic specificity, relative activity and molecular weight. This assay should be useful for many research disciplines investigating the role of lysozymes and other cell wall hydrolases capable of refolding after SDS treatment

Characterization of the SAM-key – a conserved regulatory domain of the Fun30 nucleosome remodeler

Cells need to constantly access their genetic material. However, in eukaryotic cells, DNA is compactly wrapped around nucleosomes and their presence poses a barrier for DNA transactions. To facilitate access, eukaryotes use ATP-driven molecular machines that dynamically shape chromatin structure, called nucleosome remodelers. Budding yeast Fun30 is the prototype member of the Fun30-SMARCAD1-ETL sub-family of nucleosome remodelers important for DNA repair and gene silencing. While the catalytic mechanism has been elucidated for several remodelers, for this family of single-subunit remodelers we lack mechanistic understanding. Here we report the discovery of the SAM-key, an evolutionary conserved domain with a sterile alpha motif (SAM)-like fold with one characteristic, long, protruding helix, using structure prediction, multiple sequence alignment and biochemical characterization. The SAM-key is crucial for Fun30 function, as deletion of the SAM-key from FUN30 in budding yeast leads to DNA repair and gene silencing defects similar to a deletion of FUN30. Biochemical and biophysical characterization of the SAM-key mutant in vitro showed similar folding and stability as wildtype Fun30 as well as wildtype-level binding to DNA and nucleosomes. However, the mutant is deficient in DNA-stimulated ATP hydrolysis as well as nucleosome sliding and eviction. Structure prediction using AlphaFold2 models interaction of the long helix of the SAM-key with protrusion I, a structural element of the conserved 2-lobed ATPase domain that controls catalytic activity in other remodelers. We verified the model and the interaction by crosslinking-mass spectrometry and mutation of the interface with a double point mutant Fun30-ICRR, which phenocopies the SAM-key deletion with defective ATPase activity and nucleosome remodeling. This confirms a regulatory role for the interaction of the SAM-key helix with protrusion I. Our data thereby demonstrate a central role of the SAM-key domain in mediating the activation of Fun30 catalytic activity, a new insight into the biology of this protein and highlighting the importance of allosteric activation for nucleosome remodelers

Structural characterization of Arabidopsis thaliana ethylene signaling molecules and the non-ribosomal peptide synthetase from Planktothrix agardhii

Plants employ a complex network of signaling pathways to regulate developmental processes and to mediate the responses to both environmental and biological stress factors. Ethylene is one of the key plant hormones involved in controlling this network, which has made it and its signaling pathway a target of intense research for several decades. In the model plant Arabidopsis thaliana, the plant hormone is detected by a group of five receptors (ETR1, ERS1, ETR2, ERS2 and EIN4) that resemble the sensor histidine kinases of bacterial two-component system. The main aim in this thesis study was the expression and purification of the full-length ETR1 for structural studies to gain insights into the initial steps in ethylene signaling. The FL ETR1 was successfully expressed in baculovirus expression vector system but the isolation of the receptor from the membrane was hampered. In addition to the FL ETR1, the cytosolic portion of the receptor was studied using Small Angle X-ray Scattering. The resulting SAXS model had the expected dimeric arrangement. EDR1 from A. thaliana is a CTR1-like MAPKKK that is involved in regulating disease resistance responses, cell death and also ethylene-induced senescence. It possesses an N-terminal regulatory domain and C-terminal catalytic domain wit Ser/Thr kinase activity. As EDR1 has been shown to autophosphorylate in trans, the mechanism of this was studied using X-ray crystallography. A crystal structure for the catalytically inactive kinase domain of EDR1 (EDR1-D792N) was obtained in the presence of the ATP substrate analog AMP-PNP. The asymmetric unit contained two molecules, one of which surprisingly was in an active-like conformation. Furthermore, the active-like EDR1-D792N molecule was found to form an authentic trans-autophosphorylation complex with the inactive monomer from the adjacent asymmetric unit. In addition to the plant defense signaling proteins, an adenylation (A) domain from cyanobacterial non-ribosomal peptide synthetase (NRPS) was studied. NRPSs are large multidomain enzymes that are found from a number of fungal and bacterial species and catalyze the ribosome-independent assembly of biologically active peptides with diverse composition and function. The A domain plays a central role in the NRPS system as it recognizes and activates the amino acid, which is incorporated into the growing peptide. The A domain ApnA A1 from the Anabaenopeptin synthetase of Planktothrix agardhii is an interesting member of its class as it has an unusual ability to activate two very distinct amino acids (arginine and tyrosine). Structural studies on this enzyme were performed to elucidate its bi-specificity. Based on the solved ApnA A1 structures, two active site residues with a crucial role in the substrate binding were identified. The mutation of these residues led to enzyme variants, which were mono-specific for either tyrosine or arginine, or in some instances were able to activate L-tryptophan. Additionally a number of ApnA A1 mutants were shown to activate unnatural amino acids (4-fluorophenylalanine and 4-azidophenylalanine). A final peptide product with an unnatural amino acid incorporated, could possibly have useful industrial or pharmaceutical applications

Mode of Action Study of Para-aminosalicylic Acid and Structure, Function and Inhibitor Study of the Isocitrate Dehydrogenase-2 in Mycobacterium tuberculosis

Tuberculosis (TB) killed 1.5 million people and rivaled AIDS, becoming the leading cause of death from infectious disease in 2014. The prevalence of multidrug resistant TB has intensified the current therapeutic procedure, making it urgent to find novel anti-tubercular agents and to come up with solutions to retard the emergence of the drug resistance. This dissertation focuses on the identification of drug targets, the exploration of drug resistance mechanisms, and the identification of novel inhibitors. In the first part, the mechanism of action of the classic anti-tubercular drug, para-aminosalicylic acid (PAS), was explored through genetic, cell viability and molecular modeling studies. Dihydrofolate reductase (DHFR) was identified to be the putative intracellular target of PAS. In addition, the molecular mechanism of PAS resistance was intensively investigated for the clinically relevant Rv2671 up-regulation mutant. Biochemical assays showed that Rv2671 exhibited a low DHFR activity with a high Km for the substrate, 7, 8-dihydrofolate. X-ray crystal structure of the Rv2671 in complex with NADP+ and tetrahydrofolate (THF) further confirmed the structural similarity between Rv2671 and DHFR. These studies together suggested that PAS resistance of this mutant is derived from the ability to complement the DHFR activity with the high level of Rv2671. The second part of this dissertation details the characteristics of Mycobacterium tuberculosis isocitrate dehydrogenase-2 (Mtb IDH2). The kinetic study of Mtb IDH2 suggested that it catalyzes an ordered sequential reaction by binding NADP+ first. X-ray crystal structure revealed the fairly conserved active site and dissimilar overall structure compared to human IDHs (HIDHs), suggesting a potential for drug selectivity. A screening of known inhibitors of mutant HIDHs and a high-throughput screening of Mtb whole cell active compounds were further implemented to identify inhibitors for Mtb IDH2. Two compounds from the screenings exhibited IC50s below 10 μM. The enzyme structure and the modest potency inhibitors of Mtb IDH2 can serve as viable starting points for the follow-up inhibitor development of Mtb IDH2

Mode of Action Study of Para-aminosalicylic Acid and Structure, Function and Inhibitor Study of the Isocitrate Dehydrogenase-2 in Mycobacterium tuberculosis

Tuberculosis (TB) killed 1.5 million people and rivaled AIDS, becoming the leading cause of death from infectious disease in 2014. The prevalence of multidrug resistant TB has intensified the current therapeutic procedure, making it urgent to find novel anti-tubercular agents and to come up with solutions to retard the emergence of the drug resistance. This dissertation focuses on the identification of drug targets, the exploration of drug resistance mechanisms, and the identification of novel inhibitors. In the first part, the mechanism of action of the classic anti-tubercular drug, para-aminosalicylic acid (PAS), was explored through genetic, cell viability and molecular modeling studies. Dihydrofolate reductase (DHFR) was identified to be the putative intracellular target of PAS. In addition, the molecular mechanism of PAS resistance was intensively investigated for the clinically relevant Rv2671 up-regulation mutant. Biochemical assays showed that Rv2671 exhibited a low DHFR activity with a high Km for the substrate, 7, 8-dihydrofolate. X-ray crystal structure of the Rv2671 in complex with NADP+ and tetrahydrofolate (THF) further confirmed the structural similarity between Rv2671 and DHFR. These studies together suggested that PAS resistance of this mutant is derived from the ability to complement the DHFR activity with the high level of Rv2671. The second part of this dissertation details the characteristics of Mycobacterium tuberculosis isocitrate dehydrogenase-2 (Mtb IDH2). The kinetic study of Mtb IDH2 suggested that it catalyzes an ordered sequential reaction by binding NADP+ first. X-ray crystal structure revealed the fairly conserved active site and dissimilar overall structure compared to human IDHs (HIDHs), suggesting a potential for drug selectivity. A screening of known inhibitors of mutant HIDHs and a high-throughput screening of Mtb whole cell active compounds were further implemented to identify inhibitors for Mtb IDH2. Two compounds from the screenings exhibited IC50s below 10 μM. The enzyme structure and the modest potency inhibitors of Mtb IDH2 can serve as viable starting points for the follow-up inhibitor development of Mtb IDH2