Background: Cell-to-cell transmission of α-syn via exosomes has been proposed to propagate Parkinson disease pathology.Results: Exosomes contain gangliosides, several other lipid classes, and proteins. Exosomes and ganglioside vesicles accelerate α-syn aggregation. Vesicles made of other membrane lipids do not.Conclusion: Exosomes provide catalytic environments for nucleation of α-syn aggregation.Significance: Revealing factors that promote α-syn aggregation may provide insight into Parkinson disease pathogenesis.

To compare xylans from forestry with agricultural origins, hardwood xylan (birch) and cereal arabinoxylan (rye) were hydrolyzed using two variants of the xylanase RmXyn10A, full-length enzyme and catalytic module only, from Rhodothermus marinus . Cultivations of four selected bacterial species, using the xylooligosaccharide (XOS) containing hydrolysates as carbon source, showed selective growth of Lactobacillus brevis DSMZ 1264 and Bifidobacterium adolescentis ATCC 15703. Both strains were confirmed to utilize the XOS fraction (DP 2-5), whereas putative arabinoxylooligosaccharides from the rye arabinoxylan hydrolysate were utilized by only B. adolescentis. Escherichia coli did not grow, despite its capability to grow on the monosaccharides arabinose and xylose. It was also shown that Pediococcus parvulus strain 2.6 utilized neither xylose nor XOS for growth. In summary, RmXyn10A or its catalytic module proved suitable for high-temperature hydrolysis of hardwood xylan and cereal arabinoxylan, producing XOS that could qualify as prebiotics for use in functional food products.

The insufficient operational stability of amine transaminases (ATA) constitutes a limiting factor for high productivity in chiral amine synthesis. In this work, we investigated the operational stability of a tetrameric ATA with 92% sequence identity to a Pseudomonas sp. transaminase and compared it to the two commonly used dimeric ATAs from Chromobacterium violaceum and Vibrio fluvialis. In the presence of substrate, all three ATAs featured reduced stability in comparison to their resting stability, but the tetramer showed slower inactivation rates than the dimeric ATAs. Kinetic and thermodynamic analysis revealed an amine donor induced inactivation mechanism involving accumulation of the less stable aminated enzyme−cofactor intermediate. Dissociation of the enzyme−PMP complex forms the unstable apoenzyme, which can rapidly unfold. Crystal structure analysis shed light on the structure−function relationship suggesting that the cofactor−ring binding element is stabilized in the quaternary structure conferring higher operational stability by minimizing PMP leakage and apoenzyme formation. In contrast to the common practice, increasing the amine acceptor content improved the stability and substrate turnover of dimeric ATAs. An extra supply of the pyridoxal cofactor (PLP) enhanced the stability of dimeric and tetrameric ATAs but reduced the transamination activity. The ATA inactivation mechanism described here provides valuable aspects for both process development and protein engineering.

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Amine transaminase (ATA) catalyse enantioselectively the direct amination of ketones, but insufficient stability during catalysis limits their industrial applicability. Recently, we revealed that ATAs suffer from substrate-induced inactivation mechanism involving dissociation of the enzyme-cofactor intermediate. Here, we report on engineering the cofactor-ring-binding element, which also shapes the active-site entrance. Only two point mutations in this motif improved temperature and catalytic stability in both biphasic media and organic solvent. Thermodynamic analysis revealed a higher melting point for the enzyme-cofactor intermediate. The high cofactor affinity eliminates the need for pyridoxal 5'-phosphate supply, thus making large-scale reactions more cost effective. This is the first report on stabilising a tetrameric ATA by mutating a single structural element. As this structural "hotspot" is a common feature of other transaminases it could serve as a general engineering target.

The lipid-protein film covering the interface of the lung alveolar in mammals is vital for proper lung function and its deficiency is related to a range of diseases. Here we present a molecular-level characterization of a clinical-grade porcine lung surfactant extract using a multitechnique approach consisting of 1 H-13 C solid-state nuclear magnetic spectroscopy, small-and wide-angle X-ray scattering, and mass spectrometry. The detailed characterization presented for reconstituted membranes of a lung extract demonstrates that the molecular structure of lung surfactant strongly depends on the concentration of cholesterol. If cholesterol makes up about 11% of the total dry weight of lung surfactant, the surfactant extract adopts a single liquid-ordered lamellar phase, L α(o) , at physiological temperatures. This L α(o) phase gradually changes into a liquid-disordered lamellar phase, L α(d) , when the temperature is increased by a few degrees. In the absence of cholesterol the system segregates into one lamellar gel phase and one L α(d) phase. Remarkably, it was possible to measure a large set of order parameter magnitudes |S CH | from the liquiddisordered and -ordered lamellar phases and assign them to specific C-H bonds of the phospholipids in the biological extract with no use of isotopic labeling. These findings with molecular details on lung surfactant mixtures together with the presented NMR methodology may guide further development of pulmonary surfactant pharmaceuticals that better mimic the physiological selfassembly compositions for treatment of pathological states such as respiratory distress syndrome.T he air-alveoli interface of human lungs consists of ∼100 m 2 of surface area covered by a 1-to 2-µm-thick film rich in lipids, generally referred to as lung surfactant (LS) (1, 2). The alveolar interfacial film has several important functions, including stabilizing the interface, allowing for area expansions and controlling diffusional transport (3-7). These functions strongly rely on the self-assembly structure, as well as on the mechanical, barrier, and surface properties of the dynamic LS interfacial layer, which in turn depend on its chemical composition and external conditions in terms of compression, hydration, and so on. The main components of LS are phospholipids, cholesterol, and the so-called surfactant proteins representing ∼80, 10, and 10 wt %, respectively (2).Deficiency of LS in the alveoli, or an imbalance of its chemical composition, induces a number of pathological conditions (8). For instance, neonatal respiratory distress syndrome affects prematurely born infants when LS is not yet matured or is completely lacking; this deficiency may be lethal if left untreated. In these cases, the most common treatment is intratracheal administration of an exogenous LS into the lungs (9). In case of acute respiratory distress syndrome the mortality is 35 to 45% (10) and an effective treatment is clearly lacking (8). An accurate characterization of the molecular structure and dynamics of endogenous an...

For the amine transaminase (ATA)-catalyzed synthesis of chiral amines, the choice of donor substrate is of high importance for reaction and process design. Alanine was investigated as an amine donor for the reductive amination of a poorly water-soluble ketone (4-phenyl-2-butanone) in a combined in situ product removal (ISPR) approach using liquid-membrane extraction together with an enzyme cascade. This ISPR strategy facilitates very high (>98%) product purity with an integrated enrichment step and eliminates product as well as coproduct inhibition. In the presented proof-of-concept alanine shows the following advantages over the other frequently employed amine donor isopropyl amine: (i) nonextractability of alanine affords high product purity without any additional downstream step and no losses via coextraction, (ii) higher maximum reaction rates, and (iii) broader acceptance among ATAs.

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