Identification, characterization and molecular adaptation of class I redox systems for the production of hydroxylated diterpenoids

Background De novo production of multi-hydroxylated diterpenoids is challenging due to the lack of efficient redox systems. Results In this study a new reductase/ferredoxin system from Streptomyces afghaniensis (AfR·Afx) was identified, which allowed the Escherichia coli-based production of the trihydroxylated diterpene cyclooctatin, a potent inhibitor of human lysophospholipase. This production system provides a 43-fold increase in cyclooctatin yield (15 mg/L) compared to the native producer. AfR·Afx is superior in activating the cylcooctatin-specific class I P450s CotB3/CotB4 compared to the conventional Pseudomonas putida derived PdR·Pdx model. To enhance the activity of the PdR·Pdx system, the molecular basis for these activity differences, was examined by molecular engineering. Conclusion We demonstrate that redox system engineering can boost and harmonize the catalytic efficiency of class I hydroxylase enzyme cascades. Enhancing CotB3/CotB4 activities also provided for identification of CotB3 substrate promiscuity and sinularcasbane D production, a functionalized diterpenoid originally isolated from the soft coral Sinularia sp

Characterisation of physico-mechanical properties and degradation potential of calcium alginate beads for use in embolisation

High molecular weight alginate beads with 59% mannuronic acid content or 68% guluronic acid were prepared using a droplet generator and crosslinked in calcium chloride. The alginate beads were compared to current embolisation microspheres for compressibility and monitored over 12 weeks for size and weight change at 37°C in low volumes of ringers solutions. A sheep uterine model was used to analyse bead degradation and inflammatory response over 12 weeks. Both the in vitro and in vivo data show good delivery, with a compressibility similar to current embolic beads. In vitro, swelling was noted almost immediately and after 12 weeks the first signs of degradation were noted. No difference was noted in vivo. This study has shown that high molecular weight alginate gel beads were well tolerated by the body, but beads associated with induced thrombi were susceptible to inflammatory cell infiltration. The beads were shown to be easy to handle and were still observable after 3 months in vivo. The beads were robust enough to be delivered through a 2.7 Fr microcatheter. This study has demonstrated that high molecular weight, high purity alginate bead can be considered as semi-permanent embolisation beads, with the potential to bioresorb over time

THE EUROPEAN DATA RELAY SYSTEM (EDRS): OPERATIONAL CHALLENGES

This paper will illustrate the challenges and preliminary solutions in operating the EDRS constellation. EDRS will include two communication payloads hosted on a dedicated spacecraft and as piggy-back on a commercial satellite. The two satellites will be positioned in geosynchronous orbit to provide nearglobal coverage for satellites in low earth orbit (LEO). EDRS is designed to reduce time delays in the transmission of large amounts of data and to allow faster access for end users. This is achieved by using an optical Laser Communication Terminal (LCT) for the link between the LEO and the EDRS payload and a Ka-band link between the EDRS payload and the ground. The latter will be established via three dedicated feeder link ground stations in Europe from where the data is distributed to the users. The users may also use their own ground stations directly receiving the data. By using EDRS extended capabilities for TM/TC operations will be possible with LEO satellites. This will enable short-time changes to the payload timeline and better reactions to anomalies while optimizing the number of necessary ground stations. DLR with its German Space Operations Center (GSOC) plays a major role in EDRS operations. This role includes design, development and integration of ground infrastructure and operations of the satellites and ground stations. The EDRS concept of operations di�ers from the conventional communication satellites. Two challenging new technologies will be integrated in order to provide faster data turnaround times and downlink capabilities of up to 600 Mbit/s. 1. Laser-Optical Inter-satellite link: The large distance between a satellite in GEO and one in LEO makes the pointing of both LCT very difficult. Good attitude information and control of both satellites is required. A good quality orbit determination is vital for good laser acquisition times and both payloads need to keep accurately track of their fast moving counterparts. Thus, development of the operations concept requires consideration of the interfaces and coordination of operations with the LEO satellites, which are operated by di�erent control centres. 2. Ka-band up- and downlink: The small wavelength of the Ka-band signal leads to signi�cant atmospheric and rain attenuation. Besides that, due to the sensibility of Ka-band technology the requirements for ground stations (pointing accuracy, higher doppler shifts and high data rates) are very challenging compared to standard S/X/Ku-band ground stations. Careful consideration has to be taken designing the ground stations, during link establishment and station operations

Requirements management for mission preparation at the German Space Operations Center (GSOC)

Verification of a contract consisting of a lot of free text and no precise requirements is a complex task. This paper describes the approach taken at GSOC during the preparation of a geostationary communication satellite mission to make this task transparent and efficient for both the customer and the operations provider. For the management of contractual requirements the commercial software DOORS together with a few specific customizations was used. All requirements and activities related to their fulfillment were stored in a central database and linked with each other. For that reason complete traceability from contract to subsystem level tests, operational procedures, inspections and design documents could be ensured continuously. Manual generation of verification matrices was rendered unnecessary. An extraction tool made it possible to provide relevant information whenever requested to the customer who was not using DOORS. This approach also enabled verification of internal requirements and tracking of the execution status of the planned tests without additional effort

Mixed quantum and classical simulation techniques for mapping electron transfer in proteins

El objetivo de esta tesis se centra en el estudio de la transferencia de electrones (ET), una de las reacciones más simples y cruciales en bioquímica. Para dichos procesos, obtener información directa de los factores que lo promueves, asi como del camino de transferencia electronica, no es una tarea trivial. Dicha información a un nivel de conocimiento detallado atómico y electrónico, sin embargo, es muy valiosa en términos de una mejor comprensión del ciclo enzimático, que podría conducir, por ejemplo, a un diseño más eficaz de inhibidores. El objetivo principal de esta tesis es el desarrollo de una metodología para el estudio cuantitativo de la ET en los sistemas biológicos. En este sentido, hemos desarrollado un nuevo método para obtener el camino de transferencia electrónico, llamado QM/MM e-­‐ Pathway, que se puede aplicar en sistemas complejos con ET de largo alcance. El método se basa en una búsqueda sucesiva de residuos importantes para la ET, utilizando la modificación de la región quantica en métodos mixtos QM/MM, y siguiendo la evolución de la densidad de espín dentro de la zona de transferencia. Hemos demostrado la utilidad y la aplicabilidad del algoritmo en el complejo P450cam/Pdx, identificando el papel clave de la Arg112 (en P450cam) y del Asp48 (en Pdx), ambos conocidos en la literatura. Además de obtener caminos de ET, hemos cuantificado su importancia en términos del acoplamiento electrónico entre el dador y aceptor para los diferentes caminos. En este sentido, se realizaron dos estudios de la influencia del solvente y de la temperatura en el acoplamiento electrónico para sistemas modelo oligopéptidos. Ambos estudios revelaron que los valores del acoplamiento electrónico fluctúan fuertemente a lo largo de las trayectorias de dinámica molecular obtenidas, y el mecanismo de transferencia de electrones se ve ampliamente afectado por el espacio conformacional del sistema. La combinación del QM/MM e-­‐pathway y de los cálculos de acoplamiento electronico fueron utilizados finalmente para investigar la ET en el complejo CCP/Cytc. Nuestros hallazgos indican el papel fundamental del Trp191 en localizar un estadio intermedio para la transferencia electronica, así como el camino ET principal que incluye Ala194, Ala193, Gly192 y Trp191. Ambos hallazgos fueron confirmados a través de la literatura. Los resultados obtenidos para el muestro de manios de ET, junto con su evaluación a través de cálculos de acoplamiento electrónico, sugieren un enfoque sencillo y prometedor para investigar ET de largo alcance en proteínas.The focus of this PhD thesis lies on electron transfer (ET) processes, belonging to the simplest but most crucial reactions in biochemistry. Getting direct information of the forces driving the process and the actual electron pathway is not a trivial task. Such atomic and electronic detailed information, however, is very valuable in terms of a better understanding of the enzymatic cycle, which might lead, for example, to more efficient protein inhibitor design. The main objective of this thesis was the development of a methodology for the quantitative study of ET in biological systems. In this regard, we developed a novel approach to map long-­‐range electron transfer pathways, called QM/MM e-­‐Pathway. The method is based on a successive search for important ET residues in terms of modifying the QM region following the evolution of the spin density of the electron (hole) within a given transfer region. We proved the usefulness and applicability of the algorithm on the P450cam/Pdx complex, indicating the key role of Arg112 of P450cam and Asp48 of Pdx for its ET pathway, both being known to be important from the literature. Besides only identifying the ET pathways, we further quantified their importance in terms of electronic coupling of donor and acceptor incorporating the particular pathway residues. Within this regard, we performed two systematic evaluations of the underlying reasons for the influence of solvent and temperature onto electronic coupling in oligopeptide model systems. Both studies revealed that electronic coupling values strongly fluctuate throughout the molecular dynamics trajectories obtained, and the mechanism of electron transfer is affected by the conformational space the system is able to occupy. Combining both ET mapping and electronic coupling calculations, we finally investigated the electron transfer in the CcP/Cytc complex. Our findings indicate the key role of Trp191 being the bridge-­‐localized state of the ET as well as the main pathway consisting of Ala194, Ala193, Gly192 and Trp191 between CcP and Cytc. Both findings were confirmed through the literature. Moreover, our calculations on several snapshots state a nongated ET mechanism in this protein complex. The methodology developed along this thesis, mapping ET pathways together with their evaluation through electronic coupling calculations, suggests a straightforward and promising approach to investigate long-­‐range ET in proteins

EDRS Operations at GSOC- relevant heritage and new developments

Das Paper beschreibt frühere Missionen des GSOC, die für das Projekt EDRS von Bedeutung sind und vorläufugt Entwürfe für den EDRS Betrieb

Mapping protein electron transfer pathways with QM/MM methods

Mixed quantum mechanics/molecular mechanics (QM/MM) methods offer a valuable computational tool for understanding the electron transfer pathway in protein–substrate interactions and protein–protein complexes. These hybrid methods are capable of solving the Schrödinger equation on a small subset of the protein, the quantum region, describing its electronic structure under the polarization effects of the remainder of the protein. By selectively turning on and off different residues in the quantum region, we are able to obtain the electron pathway for short- and large-range interactions. Here, we summarize recent studies involving the protein–substrate interaction in cytochrome P450 camphor, ascorbate peroxidase and cytochrome c peroxidase, and propose a novel approach for the long-range protein–protein electron transfer. The results on ascorbate peroxidase and cytochrome c peroxidase reveal the importance of the propionate groups in the electron transfer pathway. The long-range protein–protein electron transfer has been studied on the cytochrome c peroxidase–cytochrome c complex. The results indicate the importance of Phe82 and Cys81 on cytochrome c, and of Asn196, Ala194, Ala176 and His175 on cytochrome c peroxidase

In-silico Assessment of Protein-Protein Electron Transfer. A Case Study: Cytochrome c Peroxidase – Cytochrome c

<div><p>The fast development of software and hardware is notably helping in closing the gap between macroscopic and microscopic data. Using a novel theoretical strategy combining molecular dynamics simulations, conformational clustering, <i>ab-initio</i> quantum mechanics and electronic coupling calculations, we show how computational methodologies are mature enough to provide accurate atomistic details into the mechanism of electron transfer (ET) processes in complex protein systems, known to be a significant challenge. We performed a quantitative study of the ET between Cytochrome c Peroxidase and its redox partner Cytochrome c. Our results confirm the ET mechanism as hole transfer (HT) through residues Ala194, Ala193, Gly192 and Trp191 of CcP. Furthermore, our findings indicate the fine evolution of the enzyme to approach an elevated turnover rate of 5.47×10⁶ s⁻¹ for the ET between Cytc and CcP through establishment of a localized bridge state in Trp191.</p> </div

Electron transfer region of the CcP/Cytc complex.

<p>The ET pathway proposed by Pelletier and Kraut is shown in red, the ET pathway suggested by Siddarth is shown in blue.</p