An α2,3‐Sialyltransferase from Photobacterium phosphoreum with Broad Substrate Scope: Controlling Hydrolytic Activity by Directed Evolution

Defined sialoglycoconjugates are important molecular probes for studying the role of sialylated glycans in biological systems. We show that the α2,3‐sialyltransferase from Photobacterium phosphoreum JT‐ISH‐467 (2,3SiaTpph) tolerates a very broad substrate scope for modifications in the sialic acid part, including bulky amide variation, C5/C9 substitution, and C5 stereoinversion. To reduce the enzyme's hydrolytic activity, which erodes the product yield, an extensive structure‐guided mutagenesis study identified three variants that show up to five times higher catalytic efficiency for sialyltransfer, up to ten times lower efficiency for substrate hydrolysis, and drastically reduced product hydrolysis. Variant 2,3SiaTpph (A151D) displayed the best performance overall in the synthesis of the GM3 trisaccharide (α2,3‐Neu5Ac‐Lac) from lactose in a one‐pot, two‐enzyme cascade. Our study demonstrates that several complementary solutions can be found to suppress the common problem of undesired hydrolysis activity of microbial GT80 sialyltransferases. The new enzymes are powerful catalysts for the synthesis of a wide variety of complex natural and new‐to‐nature sialoconjugates for biological studies

Fermi Bubbles in the Milky Way: the closest AGN feedback laboratory courtesy of Sgr A*?

Deposition of a massive ($10^4$ to 10^5 \msun) giant molecular cloud (GMC) into the inner parsec of the Galaxy is widely believed to explain the origin of over a hundred unusually massive young stars born there $\sim 6$ Myr ago. An unknown fraction of that gas could have been accreted by Sgr A*, the supermassive black hole (SMBH) of the Milky Way. It has been recently suggested that two observed $\gamma$-ray-emitting bubbles emanating from the very center of our Galaxy were inflated by this putative activity of Sgr A*. We run a suite of numerical simulations to test whether the observed morphology of the bubbles could be due to the collimation of a wide angle outflow from Sgr A* by the disc-like Central Molecular Zone (CMZ), a well known massive repository of molecular gas in the central $\sim 200$ pc. We find that an Eddington-limited outburst of Sgr A* lasting $\simeq 1$ Myr is required to reproduce the morphology of the {\it Fermi} bubbles, suggesting that the GMC mass was \sim 10^5 \msun and it was mainly accreted by Sgr A* rather than used to make stars. We also find that the outflow from Sgr A* enforces strong angular momentum mixing in the CMZ disc, robustly sculpting it into a much narrower structure -- a ring -- perhaps synonymous with the recently reported "Herschel ring". In addition, we find that Sgr A* outflow is likely to have induced formation of massive star-forming GMCs in the CMZ. In this scenario, the Arches and Quintuplet clusters, the two observed young star clusters in the central tens of parsecs of the Galaxy, and also GMCs such as Sgr B2, owe their existence to the recent Sgr A* activity.Comment: 18 pages, 10 figures, submitted to MNRA

Evaluierung bakterieller Enzyme für die Synthese neuartiger neo-Sialokonjugate

Sialinsäuren bilden eine Familie von 9-Kohlenstoff-Zuckern mit einer α-Ketonsäurefunktionalität und sind durch ihre strukturelle Komplexität direkt mit zahlreichen biologischen Erkennungs- und Steuerungsprozessen assoziiert. Glycokonjugate, die terminale Sialinsäuren enthalten, sind omnipräsent in tierischen Glycanen wie Glycoproteinen und Glycolipiden und auf der äußersten Schicht der Zelloberfläche zu finden. Auf der anderen Seite können Sialokonjugate als biologische Maske für pathogene Bakterien fungieren, die sialinsäurehaltige Oligosaccharide produzieren, um sich vor dem Immunsystem des Wirts zu schützen oder als wirtseitige Andockstation für Viruspartikel dienen. Um die Funktionen und die biologische Bedeutung von Sialinsäuren und Sialokonjugaten in der Natur zu verstehen bzw. neuartige pharmazeutische Ansätze zu entwickeln, ist die Synthese von neuartigen neo-Sialokonjugaten ein unerlässlicher Ansatz. Um die Akzeptanz von Sialinsäurestrukturen mit strukturellen Variationen des terminalen C7–C9 Kohlenstoffrückgrats durch Enzyme des Leloir-Biosynthesewegs zu evaluieren, wurde eine Reihe neuartiger Sialinsäureanaloga synthetisiert. In einem neuartigen semisynthetischen Ansatz wurden, ausgehend von natürlicher N-Acetylneuraminsäure, über eine oxidative Spaltung des C7–C9 Rückgrats und darauffolgende Einführung nicht natürlicher Strukturmotive, chemoenzymatisch anderweitig nicht zugängliche Derivate und Analoga synthetisiert. Durch diastereoselektive Rekonstruktion des Rückgrats mittels Barbier-artiger Carboligation konnten vollständig diastereomerenreine Verbindungen mit gesättigten und ungesättigten sowie linearen und verzweigten und partiell desoxygenierten Alkylketten synthetisiert werden. Ebenso konnten Sialindisäuren und das erste verkürzte Legionaminsäureanalogon synthetisiert werden. Diese neuartigen Sialinsäuren wurden auf ihre Reaktionsfreudigkeit bezüglich der Nukleotidaktivierung durch die CMP-Sialat-Synthetase aus Neisseria meningitidis unter Verwendung eines pH-abhängigen Assays kinetisch analysiert. Über enzymatische Eintopfreaktionen wurden Nukleotidaktivierung und Sialyltransfer auf Lactose durchgeführt, um neuartige Trisaccharide als GM3 Analoga herzustellen, welche dann zur Testen der Substrattoleranz zweier bakterieller Neuraminidasen eingesetzt wurden. Für den Sialyltransfer wurde eine α2,3-Sialyltransferase aus Photobacterium phosphoreum JT-ISH-467 in einem Mutageneseansatz optimiert, um die hydrolytischen Aktivitäten dieses Enzyms durch gelenkte Evolution zu minimieren. In einer strukturbasierten Sättigungsmutagenese konnten drei Varianten mit bis zu 5-fach höherer katalytischer Effizienz für den Sialyltransfer (S359T / S360T) und bis zu 10-fach reduzierter Effizienz der Hydrolyse (L387A) gefunden werden. Als Variante mit dem effektivsten Profil wurde die Variante A151D identifiziert, die 4.3-fach reduzierter Hydrolyse und 1.9-fach gesteigerte Sialyltransfereffizienz zeigte. In einer NMR-Studie wurde schließlich bestätigt, dass die Sialidaseaktivität bei L387A bis zu 68-fach und bei A151D 26-fach reduziert ist

Semi‐Synthetic Sialic Acid Probes for Challenging the Substrate Promiscuity of Enzymes in the Sialoconjugation Pathway

A series of unusual sialic acid analogs were prepared using a semi‐synthetic strategy. Truncation of natural N‐acetylneuraminic acid was followed by diastereoselective carbon backbone reconstruction using Barbier‐type carboligations as well as different functional group interconversions, which provided access to a variety of functional motifs in the terminal carbon backbone, including examples of saturated and unsaturated, linear and branched alkyl chains, partially deoxygenated sialic acids, sialic diacids and a first truncated legionaminic acid analog. The synthetic sialic acid probes were studied for nucleotide activation by the CMP‐sialic acid synthetase from Neisseria meningitidis using a universal pH‐shift assay for kinetic analysis. One‐pot enzymatic nucleotide activation and sialyltransfer to lactose was performed using a selection of five probes together with an engineered α2,3‐sialyltransferase from Photobacterium phosphoreum to furnish five new‐to‐nature analogs of the GM3 trisaccharide, which were finally utilized to test the substrate tolerance of two bacterial sialidases. The obtained set of sialic acid analogs and neo‐sialocojugates provides interesting opportunities for further glycobiology studies

Ergodicity of perpendicular cosmic ray transport

Aims. The random walk of energetic charged particles in turbulent magnetic fields is investigated. Special focus is placed on transport across the mean magnetic field, which had been found to be subdiffusive on many occasions. Therefore, a characterization using the concept of ergodicity is attempted by noting the connection to the time evolution of the mean-square displacement. Methods. Based on the test-particle approach, a numerical Monte-Carlo simulation code is used to integrate the equation of motion for particles that are scattered by magnetic turbulence. The turbulent fields are generated by superposing plane waves with a Kolmogorov-type power spectrum. The individual particle trajectories are then used to calculate a variety of statistical quantities. Results. The simulation results clearly demonstrate how the heterogeneity of the particle ensemble causes the system to be weakly non-ergodic. In addition, it is shown how the step length distribution varies with the particle energy. In conclusion, cross-field transport is non-Gaussian but still almost diffusive.Comment: 7 pages, 8 figures, accepted for publication in Astronomy & Astrophysic

New constraints on modelling the random magnetic field of the MW

We extend the description of the isotropic and anisotropic random component of the small-scale magnetic field within the existing magnetic field model of the Milky Way from Jansson&Farrar, by including random realizations of the small-scale component. Using a magnetic-field power spectrum with Gaussian random fields, the NE2001 model for the thermal electrons and the Galactic cosmic-ray electron distribution from the current GALPROP model we derive full-sky maps for the total and polarized synchrotron intensity as well as the Faraday rotation-measure distribution. While previous work assumed that small-scale fluctuations average out along the line-of-sight or which only computed ensemble averages of random fields, we show that these fluctuations need to be carefully taken into account. Comparing with observational data we obtain not only good agreement with 408 MHz total and WMAP7 22 GHz polarized intensity emission maps, but also an improved agreement with Galactic foreground rotation-measure maps and power spectra, whose amplitude and shape strongly depend on the parameters of the random field. We demonstrate that a correlation length of ~220pc (50pc being a 5\sigma lower limit) is needed to match the slope of the observed power spectrum of Galactic foreground rotation-measure maps. Using multiple realizations allows us also to infer errors on individual observables. We find that previously-used amplitudes for random and anisotropic random magnetic field components need to be rescaled by factors of ~0.3 and 0.6 to account for the new small-scale contributions. Our model predicts a rotation measure of -2.8\pm7.1 rad/m^2 and 4.4\pm11.0 rad/m^2 for the north and south Galactic poles respectively, in good agreement with observations. Applying our model to deflections of ultra-high-energy cosmic rays we infer a mean deflection of ~3.5\pm1.1 degree for 60 EeV protons arriving from CenA.Comment: 28 pages, 12 figures, 2 table