Naphthalene dioxygenase from Pseudomonas sp. NCIB 9816-4 : systematic analysis of the active site

Die gezielte Oxyfunktionalisierung von Olefinen gehört zu den am meist gesuchten Reaktionen in der Chemie. Insbesondere die Dihydroxylierung und die daraus resultierenden chiralen, vicinalen 1,2-Diole spielen hierbei eine wichtige Rolle. So werden 1,2-Diole sowohl als chirale Liganden und Auxiliare und als chirale Synthons für Pharmabausteine sowie Agrochemikalien eingesetzt. Eine schnelle und effiziente Möglichkeit für die stereoselektive, asymmetrische Sharpless Dihydroxylierung (AD) von C=C-Doppelbindungen ergibt sich aus der Metall-katalysierten Oxyfunktionalisierung mittels Osmium oder anderen Übergangsmetallen. Neben der guten Ausbeute und der hohen Selektivität, stellen jedoch vor allem die Toxizität der Katalysatoren, sowie auch die Überoxidation und Spaltung der generierten cis-Diole Herausforderungen in der Anwendung dar. Rieske Nicht-Häm Dioxygenasen (ROs) sind eine biologische Alternative zur rein chemischen, asymmetrischen Dihydroxylierung. In der Natur sind diese Multikomponentensysteme, bestehend aus einer hexameren Oxygenase, einem Elektronen-Shuttlemolekül und einer Reduktase, für die Dihydroxylierung von aromatischen Motiven verantwortlich und katalysieren den ersten Schritt im Katabolismus von Aromaten. Mit der Entdeckung dieser effizienten Bio-katalysatoren wurde eine umweltfreundliche Alternative zur chemisch katalysierten Sharpless AD entdeckt. Aufgrund der Verfügbarkeit von Kristallstrukturen wurde die Naphthalen Dioxygenase (NDO) aus Pseudomonas sp. NCIB 9816-4 als ein Vertreter der ROs für das semi-rationale Design ausgewählt und Varianten im aktiven Zentrum des Enzyms generiert. Neben der direkten Katalyse am aromatischen Ring, wurde durch Variation der Substituenten auch die allylische Mono- bzw. die cis-Dihydroxylierung von Alkenylresten in aromatischen Molekülen (z. B. α-Methylstyrol, Allylbenzol) und die Katalyse von C=C-Doppelbindungen in nicht-aromatischen, nicht-planaren Molekülen (z. B. R-Limonen) gezeigt. Aufgrund der Vielfältigkeit dieser Enzyme besteht ein gesteigertes Interesse am biotechnologischen Einsatz, um das enorme Potential und die Vielfältigkeit des biokatalytischen Repertoires dieser Katalysatoren ausschöpfen zu können. Des Weiteren erfolgte die nähere Betrachtung der heterologen Herstellung des Biokatalystors in Escherichia coli, wobei sowohl in vitro als auch in vivo Systeme betrachtet wurden. Hierbei stand im Fall der in vitro Untersuchungen das Zusammenspiel der unterschiedlichen Komponenten des Systems, das Reaktionssetup und der Einfluss des Cosolvents im Mittelpunkt. Für das optimierte in vitro System wurden schließlich folgende Parameter definiert: (I) Verhältnis der Komponenten mit 1 μM Oxygenase, 20 μM Ferredoxin und 5 μM Reduktase, (II) das Reaktionssetup mit 2 mM Substrat in Ethanol bei 30 °C für 2 h, und (III) der Anteil des Cosolvents Ethanol mit 5 %(v/v). Ein Alanin-Scan der zwölf first shell Aminosäuren lieferte im in vitro System bereits erste Indizien für relevante Mutagenese-Hotspots mit den Positionen A206, H295, L307, G204 und V260. Im Gegensatz zum in vivo System wurde im in vitro System eine deutlich erniedrigte Aktivität gegenüber den untersuchten substituierten Aromaten detektiert, weshalb auf eine mangelnde Stabilität der Komponenten im in vitro System geschlossen wurde. Im in vivo System wurde zunächst die Optimierung der Expression forciert, wobei das entwickelte Expressions- und Biotransformationsprotokoll zu einer guten Reproduzierbarkeit in Ganzzellansätzen mit Standardabweichungen von unter 5 % geführt hat. Hierzu wurden frisch transformierte Zellen zur Anzucht (37 °C) in TB-Medium verwendet und bei Erreichen einer optischen Dichte von 0,6-0,8 mit 0,1 mM Isopropyl-β-D-thiogalactopyranosid induziert. Nach 20-stündiger Expression bei 25 °C wurden eine Zellsuspension mit 0,1 mM Kaliumphosphatpuffer (pH 7,2) und 20 mM Glucose (0,2 gBFM/mL) für Ganzzellumsätze hergestellt. Die Reaktion wurde durch Zugabe des in Ethanol gelösten Substrates gestartet und nach 20 h bei 30 °C mit der Zugabe von Lösungsmittel gestoppt. Für die in vivo Untersuchung wurde ein semi-rationaler Mutageneseansatz gewählt, indem alle first shell Aminosäuren mit Alanin, Valin und Isoleucin (36 Varianten) ausgetauscht, sowie 25 Doppelvarianten an den Positionen A206, H295 und V260 generiert wurden. Mit dieser Bibliothek erfolgte die Identifizierung von wichtigen Struktur-Funktionsbeziehungen anhand von unterschiedlich substituierten Styrolderivaten und dem Monoterpen R-Limonen. Mit dem Einbringen einer Punktmutation in der aktiven Tasche konnten deutliche Veränderungen in der Reaktions- und Substratspezifität sowie in der Regio- und Stereoselektivität (≥ 90 %) beobachtet werden, wobei die Restaktivität gegenüber dem natürlichen Substrat Naphthalen (bis > 99 %) erhalten blieb. So stellten sich die Position A206, sowie die gegenüberliegenden Positionen H295, F202, F352, V260 und L307 in der planaren, zylinderförmigen aktiven Tasche als maßgeblich für die Steuerung der Aktivität und Selektivität der NDO dar. Generell konnte eine Abnahme der Aktivität mit steigender Substituentengröße und Verzweigungsgrad (Methyl- bis Pentyl- bzw. tert-Butyl-Reste) detektiert werden. Gleichfalls konnte eine Tendenz für ungesättigte Substituenten am Aromaten beobachtet werden, wobei die Aktivität von mono- über gem-di- und trans-di-substituierte Seitenketten abnahm. Bei der Untersuchung von unterschiedlichen Methoxystyrolderivaten konnte eine gesteigerte Spezifität und Stereoselektivität (≥ 95 %ee) beobachtet werden. Neben Hydroxylierungsreaktionen wurden hierbei auch Dealkylierungsreaktionen beobachtet. Die Dihydroxylierung wurde beim Vorliegen einer zum Aromaten konjugierten C=C-Doppelbindung gegenüber der O-Demethylierung bevorzugt. Lag die C=C-Doppelbindung isoliert zum aromatischen System vor, wurde hingegen eine Präferenz für die O-Demethylierung beobachtet. Grundsätzlich hat sich die NDO als einen guten Startpunkt für die biokatalysierte, asymmetrische Dihydroxylierung erwiesen und durch die systematische Analyse der aktiven Tasche konnten essentielle Stellschrauben für die weitere Verbesserung des Katalysator identifiziert werden.The oxyfunctionalization of olefins is one of the most sought-after reactions in chemistry. Especially chiral vicinal 1,2-diols, afforded from asymmetric dihydroxylations, are often applied as chiral ligands, auxiliars or chiral synthons for pharmaceuticals and agrochemicals. In chemistry, a very powerful method for the stereoselective, asymmetric dihydroxylation (AD) of C=C double bonds is represented by the metal-catalyzed oxyfunctionalization via osmium (Sharpless AD) or other transition metals. Through extensive research in this field, high yields and excellent stereoselectivities are achieved with these catalysts. But their toxicity as well as the formation of byproducts through oxidative ring fission reactions represent major limitations in their applicability. Rieske non-heme dioxygenases (ROs) are the effective, biological alternative to the Sharpless AD. These multicomponent systems consist of a hexameric oxygenase, an electron shuttle molecule and a reductase. In nature, they dihydroxylate C=C double bonds in aromatic rings and therefore initiate degradation of arenes. Due to the availability of different crystal structures the naphthalene dioxygenase (NDO) from Pseudomonas sp. NCIB 9816-4 was representatively investigated for the ROs and mutations in the active site were examined. By varying the substitution pattern, the direct catalysis at the aromatic ring as well as the allylic mono- and cis-dihydroxylation of the alkenyl side chain of a wide range of arenes (e. g. α-methylstyrene, allylbenzene) and the catalysis of non-aromatic, non-planar molecules like R-limonene were shown. Due to the broad biocatalytically repertoire of these enzymes the application for biotechnological purposes is of peculiar interest. To have a closer look at this catalyst the heterologous in vivo and in vitro expression in Escherichia coli was examined. Hereby, the in vitro investigation was focused on the interaction of the different components of the NDO, the reaction setup and the influence of the cosolvent. After optimization of the in vitro system the following parameters were defined: (I) ratio between the components with 1 μM oxygenase, 20 μM ferredoxin and 5 μM reductase, (II) the reaction setup with 2 mM substrate in ethanol at 30 °C for 2 h, and (III) the amount of the cosolvent ethanol with 5 %(v/v). With an alanine scan of twelve first shell amino acids in the in vitro system the positions A206, H295, L307, G204 and V260 seemed to be promising candidates for mutagenesis, although the allegedly low stability of the components in the in vitro system only led to minor product formation. Through optimization of the in vivo system a protocol for the expression and whole cell biotransformation was developed, which delivered a good reproducibility with standard deviations of less than 5 %. Freshly transformed cells were cultivated in TB-medium (37°C) until an optical density of 0,6-0,8 and then induced with 0,1 mM isopropyl-β-D-thiogalactopyranoside. The following heterologous expression was performed for 20 h at 25 °C. For whole cell biotransformations the cells were resuspended in 0,1 mM potassium phosphate buffer (pH 7,2) containing 20 mM glucose (0,2 gcww/mL). Reactions were started by adding the substrate (dissolved in ethanol) and stopped with the addition of solvent after 20 h at 30 °C. By using a semi-rational design approach in the in vivo system all twelve first shell amino acid residues were exchanged with either an alanine, a valine or an isoleucine (36 single variants) and additionally, based on in silico docking of R-limonene, 25 double variants were generated by combination of the positions A206, H295 and V260. By the screening of styrene derivatives as well as the monoterpene R-limonene important structure and function relationships were identified. The introduction of a single point mutation in the active site of the NDO had a clear influence on the reaction and substrate specificity as well as on the regio- and stereoselectivity (≥ 90 %), while the residual activity towards the natural substrate naphthalene (up to > 99 %) was not affected. Determining influences on the activity and selectivity of the NDO were identified at position A206 as well as at opposite positions H295, F202, F352, V260 and L307 of the planar cylindrical active site. In general, the activity dropped with an increasing size of the substituent as well as with increasing degree of branching (methyl- to pentyl- or tert-butyl-substituents). In case of the alkenyl side chains of the arene substrates the activity decreased from mono- to gem-di- to trans-di-substituted alkenes. Further investigation of different methoxystyrene derivates, however, showed an increase in specificity and stereoselectivity (≥ 95 %ee). By converting these para-methoxy substrates not only the allylic mono- and cis-dihydroxylation reactions were observed, but also the O-dealkylation. If the C=C double bond of the side chain was conjugated to the aromatic system a preference for the dihydroxylation was discovered, while with an isolated C=C double bond the O-demethylation was detected as a major product. In terms of selectivity and specificity is the NDO a good starting point for the selective, asymmetric dihydroxylation and the systematic analysis of the active site led to the discovery of crucial set screws for futher optimization of the catalyst

Rieske non-heme dioxygenases: Versatile biocatalysts for the generation of vicinal Cis- Diols

Vicinal cis-diols are a common motif in many biologically active natural products and important intermediates in the flavor and fragrance as well as pharmaceutical industries. Most of the chemical approaches for cis-dihydroxylations are based on transition-metal catalysts like osmium and manganese, which are expensive and toxic. Hence, the enzyme-catalyzed asymmetric dihydroxylation is a powerful tool to overcome the obstacles encountered using such chemical approaches. With the Rieske non-heme dioxygenases (ROs) a promising biocatalytic alternative to generate very selectively vicinal cis-diols is at our disposal. Also known as the non-heme analog to P450 monooxygenases1, these enzymes are multicomponent systems, which only need molecular oxygen for the vicinal cis-dihydroxylation (s. Figure 1). Please click Additional Files below to see the full abstract

Biocatalyst Screening with a Twist: Application of Oxygen Sensors Integrated in Microchannels for Screening Whole Cell Biocatalyst Variants

Selective oxidative functionalization of molecules is a highly relevant and often demanding reaction in organic chemistry. The use of biocatalysts allows the stereo- and regioselective introduction of oxygen molecules in organic compounds at milder conditions and avoids the use of complex group-protection schemes and toxic compounds usually applied in conventional organic chemistry. The identification of enzymes with the adequate properties for the target reaction and/or substrate requires better and faster screening strategies. In this manuscript, a microchannel with integrated oxygen sensors was applied to the screening of wild-type and site-directed mutated variants of naphthalene dioxygenase (NDO) from Pseudomonas sp. NICB 9816-4. The oxygen sensors were used to measure the oxygen consumption rate of several variants during the conversion of styrene to 1-phenylethanediol. The oxygen consumption rate allowed the distinguishing of endogenous respiration of the cell host from the oxygen consumed in the reaction. Furthermore, it was possible to identify the higher activity and different reaction rate of two variants, relative to the wild-type NDO. The meander microchannel with integrated oxygen sensors can therefore be used as a simple and fast screening platform for the selection of dioxygenase mutants, in terms of their ability to convert styrene, and potentially in terms of substrate specificity

Systematic review of studies generating individual participant data on the efficacy of drugs for treating soil-transmitted helminthiases and the case for data-sharing

Preventive chemotherapy and transmission control (PCT) by mass drug administration is the cornerstone of the World Health Organization (WHO)’s policy to control soil-transmitted helminthiases (STHs) caused by Ascaris lumbricoides (roundworm), Trichuris trichiura (whipworm) and hookworm species (Necator americanus and Ancylostama duodenale) which affect over 1 billion people globally. Despite consensus that drug efficacies should be monitored for signs of decline that could jeopardise the effectiveness of PCT, systematic monitoring and evaluation is seldom implemented. Drug trials mostly report aggregate efficacies in groups of participants, but heterogeneities in design complicate classical meta-analyses of these data. Individual participant data (IPD) permit more detailed analysis of drug efficacies, offering increased sensitivity to identify atypical responses potentially caused by emerging drug resistance

The biospheric emergency calls for scientists to change tactics

Our current economic and political structures have an increasingly devastating impact on the Earth’s climate and ecosystems: we are facing a biospheric emergency, with catastrophic consequences for both humans and the natural world on which we depend. Life scientists – including biologists, medical scientists, psychologists and public health experts – have had a crucial role in documenting the impacts of this emergency, but they have failed to drive governments to take action in order to prevent the situation from getting worse. Here we, as members of the movement Scientist Rebellion, call on life scientists to re-embrace advocacy and activism – which were once hallmarks of academia – in order to highlight the urgency and necessity of systemic change across our societies. We particularly emphasise the need for scientists to engage in nonviolent civil resistance, a form of public engagement which has proven to be highly effective in social struggles throughout history

Herbivory on the pedunculate oak along an urbanization gradient in Europe : Effects of impervious surface, local tree cover, and insect feeding guild

Urbanization is an important driver of the diversity and abundance of tree-associated insect herbivores, but its consequences for insect herbivory are poorly understood. A likely source of variability among studies is the insufficient consideration of intraurban variability in forest cover. With the help of citizen scientists, we investigated the independent and interactive effects of local canopy cover and percentage of impervious surface on insect herbivory in the pedunculate oak (Quercus robur L.) throughout most of its geographic range in Europe. We found that the damage caused by chewing insect herbivores as well as the incidence of leaf-mining and gall-inducing herbivores consistently decreased with increasing impervious surface around focal oaks. Herbivory by chewing herbivores increased with increasing forest cover, regardless of impervious surface. In contrast, an increase in local canopy cover buffered the negative effect of impervious surface on leaf miners and strengthened its effect on gall inducers. These results show that – just like in non-urban areas – plant-herbivore interactions in cities are structured by a complex set of interacting factors. This highlights that local habitat characteristics within cities have the potential to attenuate or modify the effect of impervious surfaces on biotic interactions.Agence Nationale de la Recherche, Grant/Award Number: ANR-10--LABX-45; Fondation BNP Paribas.info:eu-repo/semantics/publishedVersio

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio

Commonly administered drug regimens from the 129 studies collecting data on the efficacy of drugs for treating ascariasis, trichuriasis and hookworm.

<p>These regimens account for the majority (89%) of IPD suitable for efficacy estimation. Other miscellaneous drugs or regimens administered include variations on the common mebendazole regimens, levamisole, diethylcarbamazine (DEC), and plant extracts tested for their anthelmintic effect. There are also some treatments which were not reported in enough detail to characterise.</p

Geographical coverage of studies collecting data on the efficacy of drugs for treating ascariasis, trichuriasis and hookworm.

<p>The 39 countries with studies generating individual participant data (IPD) suitable for estimating drug efficacy are shaded blue, with darker shades corresponding to higher estimated abundance of IPD. Five of these, with a red border, do not require preventive chemotherapy and transmission control (PCT) for soil-transmitted helminthiases [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006053#pntd.0006053.ref024" target="_blank">24</a>] (infection prevalence <20%). while the other 34 require PCT. The 69 countries shaded in yellow had no studies identified here, and have been designated by the World Health Organization as requiring PCT. The map was created using freely available country outline data from Natural Earth (naturalearthdata.com).</p

Number of cohorts and estimated number of individual participant data by key age groups from 128 of the 129 studies on the efficacy of drugs for treating ascariasis, trichuriasis and hookworm.

<p>Age groups are assigned according to reporting of the study, which does not always coincide with World Health Organization definitions.</p