Molekularbiologische und biochemische Untersuchungen zur Biosynthese von Clorobiocin in Streptomyces roseochromogenes DS 12.976

Aminocoumarin antibiotics, such as novobiocin, clorobiocin and coumermycin A1, are produced by various Streptomyces strains and are very potent against gram-positive pathogenic bacteria including methicillin-resistant Staphylococcus strains. Bacterial DNA gyrase is the target of the aminocoumarin antibiotics. Until recently, novobiocin (Albamycin®, Pharmacia-Upjohn) was licensed in the United States for the treatment of infections with gram-positive bacteria and has been shown to enhance the cytotoxic activities of the anti-tumor drugs etoposide and teniposide. So far, the therapeutic use of aminocoumarin antibiotics is limited due to their low solubility in water, toxicity in eukaryotes and poor penetration in gram-negative bacteria. Combinatorial biosynthesis may offer a chance to develop novel aminocoumarins with improved properties. The biosynthetic gene clusters of novobiocin and coumermycin A1 were already sequenced in our laboratory. The first task of my thesis was to identify the biosynthetic gene cluster of the third "classical" aminocoumarin antibiotic, clorobiocin. The cluster was cloned by screening a cosmid library of Streptomyces roseochromogenes DS 12.976 with two heterologous probes from the novobiocin biosynthetic gene cluster. Sequence analysis revealed 29 open reading frames with striking similarity to the biosynthetic gene clusters of novobiocin and coumermycin A1. A comparison of the gene clusters of clorobiocin, novobiocin and coumermycin A1 showed that the structural differences between the three antibiotics were remarkably well reflected by differences in the organization of the biosynthetic gene clusters. The second part of my thesis was to elucidate the biosynthesis of 3-dimethylallyl-4-hydroxybenzoate moiety (Ring A) of clorobiocin and novobiocin by biochemical and molecular biological studies. Comparison of the three aminocoumarin clusters allowed us to identify three genes in the novobiocin and clorobiocin clusters for which no homologues existed in the coumermycin cluster. We speculated that these genes might be involved in the biosynthesis of Ring A (which is absent in coumermycin A1). These genes were: a) cloR and novR, which showed sequence similarity to putative aldolases; b) cloF and novF, which showed sequence similarities to dehydrogenases; c) cloQ and novQ, which did not show sequence similarities to known genes in the database. A biosynthetic pathway for clorobiocin was proposed in which activated ß-hydroxytyrosine was a common intermediate in the formation of the aminocoumarin ring (Ring B) and Ring A (Fig. 7, p. 40). However, analysis of S. roseochromogenes mutants (cloI- mutant, cloQ- mutant and cloR- mutant) revealed that Ring A and Ring B are formed by two distinct and independent pathways and that cloQ and cloR are essential for the formation of Ring A. CloQ was expressed in E. coli, purified and identified as an aromatic prenyltransferase. It is a soluble, monomeric, 35 kDa protein. 4-Hydroxyphenylpyruvate (4HPP) and dimethylallyl diphosphate (DMAPP) were identified as the substrates of this enzyme, with Km values determined as 25 and 35 µM, respectively. CloQ was found to be dissimilar from most prenyltransferases described so far and may indicate the existence of a new class of prenyltransferases. CloR was expressed in E. coli, purified and identified as a bifunctional non-heme iron oxygenase. It is a soluble, tetrameric protein. CloR converts 3-dimethylallyl-4-hydroxyphenylpyruvate via 3-dimethylallyl-4-hydroxymandelic acid (3DMA-4HMA) to Ring A. Therefore it catalyzes two consecutive oxidative decarboxylation steps. 18O2 labelling experiments showed that two oxygen atoms are incorporated into the intermediate 3DMA-4HMA in the first reaction step, but only one further oxygen is incorporated into the final product Ring A during the second reaction step. CloR does not show sequence similarity to known oxygenases. It apparently presents a novel member of the diverse family of the non-heme iron (II) and a-keto dependent oxygenases, with 3DMA-4HPP functioning both as a-ketoacid and as hydroxylation substrate. The reaction catalyzed by CloR represents a new pathway to benzoic acids in nature. In the third part of my thesis, a novO- mutant was created from the novobiocin producer S. spheroides, using a new inactivation method: the PCR targeting system. This mutant produced a derivative of novobiocin lacking the methyl group on the aminocoumarin ring.This provided functional proof for the role of novO in novobiocin biosynthesis. The mutant will be used for further experiments in combinatorial biosynthesis.Aminocoumarin-Antibiotika wie Novobiocin, Clorobiocin und Coumermycin A1 werden von verschiedenen Streptomycetenstämmen produziert und besitzen große Wirksamkeit gegenüber grampositiven pathogenen Bakterien, einschließlich Methicillin-resistenter Staphylococcusstämme. Das Zielmolekül der Aminocoumarin-Antibiotika ist die bakterielle DNA-Gyrase. Bis vor kurzen wurde Novobiocin (Albamycin®, Pharmacia-Upjohn) in den Vereinigten Staaten zur Behandlung von Infektionen mit grampositiven Bakterien eingesetzt. Es konnte gezeigt werden, dass es die cytotoxische Aktivität von Cytostatika wie Etoposid und Teniposid verstärkt. Aufgrund ihrer schlechten Wasserlöslichkeit, ihrer Toxizität für Eukaryoten und ihrer schlechten Aufnahme in gramnegative Bakterien ist der therapeutische Einsatz der Aminocoumarin-Antibiotika bislang stark eingeschränkt. Kombinatorische Biosynthese könnte eine Möglichkeit zur Entwicklung neuer Aminocoumarine mit verbesserten Eigenschaften darstellen. Voraussetzung dafür ist die genaue Kenntnis der Biosynthese der betreffenden Antibiotika. Die Biosynthesegencluster von Novobiocin und Coumermycin A1 waren bereits in unserem Labor sequenziert worden. Die erste Aufgabe meiner Doktorarbeit war die Identifizierung des Biosynthesegenclusters des dritten "klassischen" Aminocoumarin-Antibiotikums Clorobiocin. Das Cluster wurde durch Screening einer Cosmidbank von Streptomyces roseochromogenes DS 12.976 mit zwei heterologen Sonden des Novobiocin-Biosynthesegenclusters kloniert. Vergleichende Sequenzanalyse ergab 29 offene Leserahmen mit deutlicher Übereinstimmung zu den Biosynthesegenclustern von Novobiocin und Coumermycin A1. Ein Vergleich der Gencluster von Clorobiocin, Novobiocin und Coumermycin A1 zeigte, dass sich die strukturellen Unterschiede zwischen den drei Antibiotika erstaunlich genau in der Organisation der Cluster widerspiegeln. Der zweite Teil meiner Arbeit bestand in der Aufklärung der Biosynthese der 3-Dimethylallyl-4-hydroxybenzoat Gruppe (Ring A) von Clorobiocin und Novobiocin, sowohl durch biochemische als auch durch molekularbiologische Untersuchungen. Der Vergleich der drei Aminocoumarin-Cluster ermöglichte uns die Identifizierung von drei Gene, die sowohl im Novobiocin- als auch im Clorobiocin-Cluster vorkommen, für die aber keine Homologe im Coumermycin A1-Cluster existieren. Wir vermuteten, dass diese Gene an der Biosynthese von Ring A beteiligt sein könnten, da dieser in Coumermycin A1 nicht vorhanden ist. Diese Gene sind: a) cloR und novR, die Sequenzähnlichkeit zu putativen Aldolasen zeigten; b) cloF und novF, die Sequenzähnlichkeit zu Dehydrogenasen zeigten; und c) cloQ und novQ, welche keinerlei Sequenzähnlichkeit zu bekannten Genen in den Datenbanken aufwiesen. Es wurde ein Weg für die Biosynthese von Clorobiocin vorgeschlagen demzufolge aktiviertes ß-Hydroxytyrosin als gemeinsames Zwischenprodukt bei der Bildung des Aminocoumarinrings (Ring B) und des Ring A fungiert (Fig. 7, p. 40). Es konnte jedoch durch die Herstellung und Analyse von S. roseochromogenes Mutanten (cloI-, cloQ-, und cloR-) gezeigt werden, dass Ring A und Ring B auf zwei verschiedenen, unabhängigen Wegen gebildet werden. CloQ wurde in E. coli exprimiert und gereinigt und als eine aromatische Prenyltransferase identifiziert. CloQ ist ein lösliches, monomeres, 35 kD Protein. 4-Hydroxyphenylpyruvat (4HPP) und Dimethylallyldiphosphat (DMAPP) wurden als Substrate dieses Enzyms identifiziert und die Km-Werte mit 25 µM bzw. 35 µM bestimmt. Wir konnten zeigen, dass CloQ sich von den meisten bisher bekannten Prenyltransferasen unterscheidet und möglicherweise einer neuen Klasse von Prenyltransferasen angehört. CloR wurde in E. coli exprimiert und gereinigt und als eine bifunktionale non-Häm Eisen-Oxygenase identifiziert. Es ist ein lösliches, tetrameres Protein. CloR setzt 3-Dimethylallyl-4-hydroxyphenylpyruvat über 3-Dimethylallyl-4-hydroxymandelsäure zu Ring A um. Dies erfolgt in zwei aufeinanderfolgenden Decarboxylierungsschritten. 18O2-Markierungsexperimente zeigten, dass im ersten Reaktionsschritt zwei Sauerstoffatome in das 3DMA-4HMA Zwischenprodukt eingebaut werden, wohingegen aber im zweiten Schritt nur ein weiteres Sauerstoffatom in das Ring A Endprodukt inkorporiert wird. CloR zeigt keine Sequenzähnlichkeit zu bekannten Oxygenasen. Anscheinend stellt es ein neues Mitglied der divergenten Familie der non-Häm Eisen (II) und a-Ketosäure-abhängigen Oxygenasen dar, bei dem 3DMA-4HPP sowohl als a-Ketosäure als auch als Hydroxylierungssubstrat fungiert. Die von CloR katalysierte Reaktion stellt einen neuen Weg zur Biosynthese von Benzoesäuren dar

Dual role for ubiquitin in plant steroid hormone receptor endocytosis

Brassinosteroids are plant steroid hormones that control many aspects of plant growth and development, and are perceived at the cell surface by the plasma membrane-localized receptor kinase BRI1. Here we show that BRI1 is post-translationally modified by K63 polyubiquitin chains in vivo. Using both artificial ubiquitination of BRI1 and generation of an ubiquitination-defective BRI1 mutant form, we demonstrate that ubiquitination promotes BRI1 internalization from the cell surface and is essential for its recognition at the trans-Golgi network/early endosomes (TGN/EE) for vacuolar targeting. Finally, we demonstrate that the control of BRI1 protein dynamics by ubiquitination is an important control mechanism for brassinosteroid responses in plants. Altogether, our results identify ubiquitination and K63-linked polyubiquitin chain formation as a dual targeting signal for BRI1 internalization and sorting along the endocytic pathway, and highlight its role in hormonally controlled plant development

Evolution of the Chalcone Isomerase Fold from Fatty Acid-Binding to Stereospecific Enzyme

Specialized metabolic enzymes biosynthesize chemicals of ecological importance, often sharing a pedigree with primary metabolic enzymes1. However, the lineage of the enzyme chalcone isomerase (CHI) remained a quandary. In vascular plants, CHI-catalyzed conversion of chalcones to chiral (S)-flavanones is a committed step in the production of plant flavonoids, compounds that contribute to attraction, defense2, and development3. CHI operates near the diffusion limit with stereospecific control4,5. While associated primarily with plants, the CHI-fold occurs in several other eukaryotic lineages and in some bacteria. Here we report crystal structures, ligand-binding properties, and in vivo functional characterization of a non-catalytic CHI-fold family from plants. A. thaliana contains five actively transcribed CHI-fold genes, three of which additionally encode amino-terminal chloroplast-transit sequences (cTP). These three CHI-fold proteins localize to plastids, the site of de novo fatty acid (FA) biosynthesis in plant cells. Furthermore, their expression profiles correlate with those of core FA biosynthetic enzymes, with maximal expression occurring in seeds and coinciding with increased FA storage in the developing embryo. In vitro, these proteins are Fatty Acid-binding Proteins (FAP). FAP knockout A. thaliana plants exhibit elevated alpha-linolenic acid levels and marked reproductive defects, including aberrant seed formation. Notably, the FAP discovery defines the adaptive evolution of a stereospecific and catalytically ‘perfected’ enzyme6 from a non-enzymatic ancestor over a defined period of plant evolution

Lansoprazole is an antituberculous prodrug targeting cytochrome bc1

Better antibiotics capable of killing multi-drug-resistant Mycobacterium tuberculosis are urgently needed. Despite extensive drug discovery efforts, only a few promising candidates are on the horizon and alternative screening protocols are required. Here, by testing a panel of FDA-approved drugs in a host cell-based assay, we show that the blockbuster drug lansoprazole (Prevacid), a gastric proton-pump inhibitor, has intracellular activity against M. tuberculosis. Ex vivo pharmacokinetics and target identification studies reveal that lansoprazole kills M. tuberculosis by targeting its cytochrome bc(1) complex through intracellular sulfoxide reduction to lansoprazole sulfide. This novel class of cytochrome bc(1) inhibitors is highly active against drug-resistant clinical isolates and spares the human H+K+-ATPase thus providing excellent opportunities for targeting the major pathogen M. tuberculosis. Our finding provides proof of concept for hit expansion by metabolic activation, a powerful tool for antibiotic screens

Directed evolution of the suicide protein O⁶-alkylguanine-DNA alkyltransferase for increased reactivity results in an alkylated protein with exceptional stability

Here we present a biophysical, structural, and computational analysis of the directed evolution of the human DNA repair protein O-6-alkylguanine-DNA alkyltransferase (hAGT) into SNAP-tag, a self-labeling protein tag. Evolution of hAGT led not only to increased protein activity but also to that the reactivity of the suicide enzyme can be influenced by higher stability, especially of the alkylated protein, suggesting stabilizing the product of the irreversible reaction. Whereas wild-type hAGT is rapidly degraded in cells after alkyl transfer, the high stability of benzylated SNAP-tag prevents proteolytic degradation. Our data indicate that the intrinstic stability of a key a helix is an important factor in triggering the unfolding and degradation of wild-type hAGT upon alkyl transfer, providing new insights into the structure-function relationship of the DNA repair protein

A rheostat mechanism governs the bifurcation of carbon flux in mycobacteria

Fatty acid metabolism is an important feature of the pathogenicity of Mycobacterium tuberculosis during infection. Consumption of fatty acids requires regulation of carbon flux bifurcation between the oxidative TCA cycle and the glyoxylate shunt. In Escherichia coli, flux bifurcation is regulated by phosphorylation-mediated inhibition of isocitrate dehydrogenase (ICD), a paradigmatic example of post-translational mechanisms governing metabolic fluxes. Here, we demonstrate that, in contrast to E. coli, carbon flux bifurcation in mycobacteria is regulated not by phosphorylation but through metabolic cross-activation of ICD by glyoxylate, which is produced by the glyoxylate shunt enzyme isocitrate lyase (ICL). This regulatory circuit maintains stable partitioning of fluxes, thus ensuring a balance between anaplerosis, energy production, and precursor biosynthesis. The rheostat-like mechanism of metabolite-mediated control of flux partitioning demonstrates the importance of allosteric regulation during metabolic steady-state. The sensitivity of this regulatory mechanism to perturbations presents a potentially attractive target for chemotherapy

Benzothiazinones Are Suicide Inhibitors of Mycobacterial Decaprenylphosphoryl-β-

Benzothiazinones (BTZs) are antituberculosis drug candidates with nanomolar bactericidal activity against tubercle bacilli. Here we demonstrate that BTZs are suicide substrates of the FAD-dependent decaprenylphosphoryl-beta-D-ribofuranose 2'-oxidase DprE1, an enzyme involved in cell-wall biogenesis. BTZs are reduced by DprE1 to an electrophile, which then reacts in a near-quantitative manner with an active-site cysteine of DprE1, thus providing a rationale for the extraordinary potency of BTZs. Mutant DprE1 enzymes from BTZ-resistant strains reduce BTZs to inert metabolites while avoiding covalent inactivation. Our results explain the basis for drug sensitivity and resistance to an exceptionally potent class of antituberculosis agents

Changes in SARS-CoV-2 Spike versus Nucleoprotein Antibody Responses Impact the Estimates of Infections in Population-Based Seroprevalence Studies

SARS-CoV-2-specific antibody responses to the Spike (S) protein monomer, S protein native trimeric form or the nucleocapsid (N) proteins were evaluated in cohorts of individuals with acute infection (n=93) and in individuals enrolled in a post-infection seroprevalence population study (n=578) in Switzerland. Commercial assays specific for the S1 monomer, for the N protein and a newly developed Luminex assay using the S protein trimer were found to be equally sensitive in antibody detection in the acute infection phase samples. Interestingly, as compared to anti-S antibody responses, those against the N protein appear to wane in the post-infection cohort. Seroprevalence in a 'positive patient contacts' group (n=177) was underestimated by N protein assays by 10.9 to 32.2% and the 'random selected' general population group (n=311) was reduced up to 45% reduction relative to S protein assays. The overall reduction in seroprevalence targeting only anti-N antibodies for the total cohort ranged from 9.4 to 31%. Of note, the use of the S protein in its native trimer form was significantly more sensitive as compared to monomeric S proteins. These results indicate that the assessment of anti-S IgG antibody responses against the native trimeric S protein should be implemented to estimate SARS-CoV-2 infections in population-based seroprevalence studies.IMPORTANCE In the present study, we have determined SARS-CoV-2-specific antibody responses in sera of acute and post-infection phase subjects. Our results indicate that antibody responses against viral S and N proteins were equally sensitive in the acute phase of infection but that responses against N appear to wane in the post-infection phase while those against S protein persist over time. The most sensitive serological assay in both acute and post-infection phases used the native S protein trimer as binding antigen that has significantly greater conformational epitopes for antibody binding compared to the S1 monomer protein used in other assays. We believe that these results are extremely important in order to generate correct estimates of SARS-CoV-2 infections in the general population. Furthermore, the assessment of antibody responses against the trimeric S protein will be critical to evaluate the durability of the antibody response and for the characterization of a vaccine-induced antibody response

A highly potent antibody effective against SARS-CoV-2 variants of concern.

Control of the ongoing SARS-CoV-2 pandemic is endangered by the emergence of viral variants with increased transmission efficiency, resistance to marketed therapeutic antibodies, and reduced sensitivity to vaccine-induced immunity. Here, we screen B cells from COVID-19 donors and identify P5C3, a highly potent and broadly neutralizing monoclonal antibody with picomolar neutralizing activity against all SARS-CoV-2 variants of concern (VOCs) identified to date. Structural characterization of P5C3 Fab in complex with the spike demonstrates a neutralizing activity defined by a large buried surface area, highly overlapping with the receptor-binding domain (RBD) surface necessary for ACE2 interaction. We further demonstrate that P5C3 shows complete prophylactic protection in the SARS-CoV-2-infected hamster challenge model. These results indicate that P5C3 opens exciting perspectives either as a prophylactic agent in immunocompromised individuals with poor response to vaccination or as combination therapy in SARS-CoV-2-infected individuals

Towards a new tuberculosis drug: pyridomycin - nature's isoniazid

Tuberculosis, a global threat to public health, is becoming untreatable due to widespread drug resistance to frontline drugs such as the InhA-inhibitor isoniazid. Historically, by inhibiting highly vulnerable targets, natural products have been an important source of antibiotics including potent anti-tuberculosis agents. Here, we describe pyridomycin, a compound produced by Dactylosporangium fulvum with specific cidal activity against mycobacteria. By selecting pyridomycin-resistant mutants of Mycobacterium tuberculosis, whole-genome sequencing and genetic validation, we identified the NADH-dependent enoyl(Acyl-Carrier-Protein) reductase InhA as the principal target and demonstrate that pyridomycin inhibits mycolic acid synthesis in M. tuberculosis. Furthermore, biochemical and structural studies show that pyridomycin inhibits InhA directly as a competitive inhibitor of the NADH-binding site, thereby identifying a new, druggable pocket in InhA. Importantly, the most frequently encountered isoniazid-resistant clinical isolates remain fully susceptible to pyridomycin, thus opening new avenues for drug development