Structural and biochemical characterization of biotechnologically relevant enzymes

Climate change, antibiotic resistances and environmental pollution are growing threats. Therefore, finding alternatives for fossil resources and discovery of new pharmaceuticals grows more important every day. Natural compounds and their in vivo production pathways proved to be a possible solution to overcome those problems. Optimized microbial hosts can serve as sustainable production platforms for various compounds as it is done for penicillin since many years. The first research topic of this thesis are borneol dehydrogenases, enzymes which convert borneol to camphor. Enantiomerically pure camphor has numerous applications in cosmetic, pharmaceutical, and chemical industry. Thus, enantioselective borneol dehydrogenases would be an attractive candidate to achieve enantiomerically pure camphor. To better understand the differences of enantioselective and unselective borneol dehydrogenases we solved the structures of two selective borneol dehydrogenases from Salvia rosmarinus and Salvia officinalis using X-ray crystallography and cryo-electron microscopy. The obtained structures were compared to the previously solved structure of the unselective borneol dehydrogenase of Pseudomonas sp. TCUHL1. The second focus of this thesis are terpene synthases, a class of enzymes responsible for the cyclization of linear terpene precursors. The products of terpene synthases are interesting candidates for the chemical and pharmaceutical industry due to their diverse characteristics and properties. Latest advances in genome sequencing enabled the discovery of many new and diverse terpene synthases from various organisms. We report on the discovery of two terpene synthases from Coniophora. puteana, Copu5 and Copu9, that not only have identical product profiles, but also show high yields in an optimized Escherichia coli strain. Main product of both enzymes is (+)-δ-cadinol that has been shown to have cytotoxic effect on MCF7 cells and could be used as a new and sustainable anti-tumor drug. To investigate their properties and gain deeper understanding into their function, we attempted to crystallize and biochemically characterize Copu5 and Copu9.Klimawandel, Antibiotikaresistenzen und Umweltverschmutzung sind wachsende Bedrohungen. Daher wird die Suche nach Alternativen für fossile Ressourcen und die Entdeckung neuer Arzneimittel von Tag zu Tag wichtiger. Naturstoffe und ihre in-vivo- Produktionswege bieten eine mögliche Lösung dieser Probleme. Optimierte mikrobielle Wirte können als nachhaltige Produktionsplattformen für verschiedene chemische Verbindungen dienen, wie es seit vielen Jahren für Penicillin üblich ist. Der erste Fokus dieser Arbeit sind Terpensynthasen, eine Klasse von Enzymen, die für die Zyklisierung von linearen Terpenvorläufern verantwortlich sind. Die Produkte der Terpensynthasen sind aufgrund ihrer vielfältigen Eigenschaften interessante Kandidaten für die chemische und pharmazeutische Industrie. Jüngste Fortschritte in der Genomsequenzierung ermöglichten die Entdeckung vieler neuer und vielfältiger Terpensynthasen aus verschiedenen Organismen. Wir berichten über die Entdeckung zweier Terpensynthasen aus Coniophora puteana, Copu5 und Copu9, die nicht nur identische Produktprofile aufweisen, sondern auch hohe Ausbeuten in einem optimierten Escherichia coli-Stamm zeigen. Hauptprodukt beider Enzyme ist (+)-δ- Cadinol, das nachweislich eine zytotoxische Wirkung auf MCF7-Zellen hat und als neues und nachhaltiges Antitumormittel eingesetzt werden könnte. Zur Untersuchung ihrer Eigenschaften und um ein tieferes Verständnis ihrer Funktion zu erlangen, haben wir versucht, Copu5 und Copu9 zu kristallisieren und biochemisch zu charakterisieren. Der zweite Schwerpunkt dieser Arbeit sind Borneoldehydrogenasen, Enzyme, die Borneol zu Kampfer umwandeln. Enantiomerenreiner Kampfer hat zahlreiche Anwendungen in der kosmetischen, pharmazeutischen und chemischen Industrie. Daher wären enantioselektive Borneoldehydrogenasen ein attraktiver Kandidat zur Herstellung von enantiomerenreinem Kampfer. Um die Unterschiede zwischen enantioselektiven und unselektiven Borneoldehydrogenasen besser zu verstehen, haben wir die Strukturen zweier selektiver Borneoldehydrogenasen aus Salvia rosmarinus und Salvia officinalis mittels Röntgenkristallographie und Kryo-Elektronenmikroskopie gelöst. Die erhaltenen Strukturen wurden mit der zuvor gelösten Struktur der unselektiven Borneol- Dehydrogenase von Pseudomonas sp. TCU-HL1 verglichen

Towards an understanding of oleate hydratases and their application in industrial processes

Fatty acid hydratases are unique to microorganisms. Their native function is the oxidation of unsaturated C–C bonds to enable detoxification of environmental toxins. Within this enzyme family, the oleate hydratases (Ohys), which catalyze the hydroxylation of oleic acid to 10-(R)-hydroxy stearic acid (10-HSA) have recently gained particular industrial interest. 10-HSA is considered to be a replacement for 12-(R)-hydroxy stearic acid (12-HSA), which has a broad application in the chemical and pharmaceutical industry. As 12-HSA is obtained through an energy consuming synthesis process, the biotechnological route for sustainable 10-HSA production is of significant industrial interest. All Ohys identified to date have a non-redox active FAD bound in their active site. Ohys can be divided in several subfamilies, that differ in their oligomerization state and the decoration with amino acids in their active sites. The latter observation indicates a different reaction mechanism across those subfamilies. Despite intensive biotechnological, biochemical and structural investigations, surprising little is known about substrate binding and the reaction mechanism of this enzyme family. This review, summarizes our current understanding of Ohys with a focus on sustainable biotransformation

amda 2 13 a major update for automated cross platform microarray data analysis

Microarray platforms require analytical pipelines with modules for data pre-processing including data normalization, statistical analysis for identification of differentially expressed genes, cluster analysis, and functional annotation. We previously developed the Automated Microarray Data Analysis (AMDA, version 2.3.5) pipeline to process Affymetrix 3′ IVT GeneChips. The availability of newer technologies that demand open-source tools for microarray data analysis has impelled us to develop an updated multi-platform version, AMDA 2.13. It includes additional quality control metrics, annotation-driven (annotation grade of Affymetrix NetAffx) and signal-driven (Inter-Quartile Range) gene filtering, and approaches to experimental design. To enhance understanding of biological data, differentially expressed genes have been mapped into KEGG pathways. Finally, a more stable and user-friendly interface was designed to integrate the requirements for different platforms. AMDA 2.13 allows the analysis of Affymetrix..

Gene amplifications cause high-level resistance against albicidin in gram-negative bacteria

Antibiotic resistance is a continuously increasing concern for public healthcare. Understanding resistance mechanisms and their emergence is crucial for the development of new antibiotics and their effective use. The peptide antibiotic albicidin is such a promising candidate that, as a gyrase poison, shows bactericidal activity against a wide range of gram-positive and gram-negative bacteria. Here, we report the discovery of a gene amplification–based mechanism that imparts an up to 1000-fold increase in resistance levels against albicidin. RNA sequencing and proteomics data show that this novel mechanism protects Salmonella Typhimurium and Escherichia coli by increasing the copy number of STM3175 (YgiV), a transcription regulator with a GyrI-like small molecule binding domain that traps albicidin with high affinity. X-ray crystallography and molecular docking reveal a new conserved motif in the binding groove of the GyrI-like domain that can interact with aromatic building blocks of albicidin. Phylogenetic studies suggest that this resistance mechanism is ubiquitous in gram-negative bacteria, and our experiments confirm that STM3175 homologs can confer resistance in pathogens such as Vibrio vulnificus and Pseudomonas aeruginosa

A New Thiopurine S-Methyltransferase Haplotype Associated With Intolerance to Azathioprine

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Branch point strength controls species-specific CAMK2B alternative splicing and regulates LTP

Regulation and functionality of species-specific alternative splicing has remained enigmatic to the present date. Calcium/calmodulin-dependent protein kinase IIβ (CaMKIIβ) is expressed in several splice variants and plays a key role in learning and memory. Here, we identify and characterize several primate-specific CAMK2B splice isoforms, which show altered kinetic properties and changes in substrate specificity. Furthermore, we demonstrate that primate-specific CAMK2B alternative splicing is achieved through branch point weakening during evolution. We show that reducing branch point and splice site strengths during evolution globally renders constitutive exons alternative, thus providing novel mechanistic insight into cis-directed species-specific alternative splicing regulation. Using CRISPR/Cas9, we introduce a weaker, human branch point sequence into the mouse genome, resulting in strongly altered Camk2b splicing in the brains of mutant mice. We observe a strong impairment of long-term potentiation in CA3-CA1 synapses of mutant mice, thus connecting branch point–controlled CAMK2B alternative splicing with a fundamental function in learning and memory

The SIB Swiss Institute of Bioinformatics' resources: focus on curated databases

The SIB Swiss Institute of Bioinformatics (www.isb-sib.ch) provides world-class bioinformatics databases, software tools, services and training to the international life science community in academia and industry. These solutions allow life scientists to turn the exponentially growing amount of data into knowledge. Here, we provide an overview of SIB's resources and competence areas, with a strong focus on curated databases and SIB's most popular and widely used resources. In particular, SIB's Bioinformatics resource portal ExPASy features over 150 resources, including UniProtKB/Swiss-Prot, ENZYME, PROSITE, neXtProt, STRING, UniCarbKB, SugarBindDB, SwissRegulon, EPD, arrayMap, Bgee, SWISS-MODEL Repository, OMA, OrthoDB and other databases, which are briefly described in this article

Structural and biochemical studies of biotechnologically relevant enzymes

Climate change, antibiotic resistances and environmental pollution are growing threats. Therefore, finding alternatives for fossil resources and discovery of new pharmaceuticals grows more important every day. Natural compounds and their in vivo production pathways proved to be a possible solution to overcome those problems. Optimized microbial hosts can serve as sustainable production platforms for various compounds as it is done for penicillin since many years. The first research topic of this thesis are borneol dehydrogenases, enzymes which convert borneol to camphor. Enantiomerically pure camphor has numerous applications in cosmetic, pharmaceutical, and chemical industry. Thus, enantioselective borneol dehydrogenases would be an attractive candidate to achieve enantiomerically pure camphor. To better understand the differences of enantioselective and unselective borneol dehydrogenases we solved the structures of two selective borneol dehydrogenases from Salvia rosmarinus and Salvia officinalis using X-ray crystallography and cryo-electron microscopy. The obtained structures were compared to the previously solved structure of the unselective borneol dehydrogenase of Pseudomonas sp. TCUHL1. The second focus of this thesis are terpene synthases, a class of enzymes responsible for the cyclization of linear terpene precursors. The products of terpene synthases are interesting candidates for the chemical and pharmaceutical industry due to their diverse characteristics and properties. Latest advances in genome sequencing enabled the discovery of many new and diverse terpene synthases from various organisms. We report on the discovery of two terpene synthases from Coniophora. puteana, Copu5 and Copu9, that not only have identical product profiles, but also show high yields in an optimized Escherichia coli strain. Main product of both enzymes is (+)-δ-cadinol that has been shown to have cytotoxic effect on MCF7 cells and could be used as a new and sustainable anti-tumor drug. To investigate their properties and gain deeper understanding into their function, we attempted to crystallize and biochemically characterize Copu5 and Copu9

Fluorine-induced polarity increases inhibitory activity of BPTI towards chymotrypsin

Substituting the P1 position in bovine pancreatic trypsin inhibitor (BPTI) is known to heavily influence its inhibitory activity towards serine proteases. Side-chain fluorinated aliphatic amino acids have been shown to alter numerous properties of peptides and proteins and thus are of interest in the context of BPTI. In our study, we systematically investigated the site-specific incorporation of non-canonical amino acids into BPTI by microwave-assisted solid-phase peptide synthesis (SPPS). Inhibitor activity of the variants was tested towards the serine protease α-chymotrypsin. We observed enhanced inhibition of two fluorinated BPTIs compared to wild type and hydrocarbon variants. To further investigate the complexes, we performed X-ray structure analysis. Our findings underline the power fluorine offers as a tool in protein engineering to beneficially alter the effects on phenomena as protein–protein interactions