Omanische Heilpflanze im Fokus der Phytochemie IPB-Wissenschaftler und Partner aus Dhofar haben jüngst die omanische Heilpflanze Terminalia dhofarica unter die phytochemische Lupe genommen. Die Pflanze ist reich an…
Geschmack ist vorhersagbar: Mit FlavorMiner. FlavorMiner heißt das Tool, das IPB-Chemiker und Partner aus Kolumbien jüngst entwickelt haben. Das Programm kann, basierend auf maschinellem Lernen (KI), anhand der…
Microbial transglutaminase (MTG, EC 2.3.2.13) of Streptomyces mobaraensis is widely used in industry for its ability to synthesize isopeptide bonds between the proteinogenic side chains of glutamine and lysine. The activated wild-type enzyme irreversibly denatures at 60 °C with a pseudo-first-order kinetics and a half-life time (t1/2) of 2 min. To increase the thermoresistance of MTG for higher temperature applications, we generated 31 variants based on previous results obtained by random mutagenesis, DNA shuffling and saturation mutagenesis. The best variant TG16 with a specific combination of five of seven substitutions (S2P, S23Y, S24 N, H289Y, K294L) shows a 19-fold increased half-life at 60 °C (t1/2 = 38 min). As measured by differential scanning fluorimetry, the transition point of thermal unfolding was increased by 7.9 °C. Also for the thermoresistant variants, it was shown that inactivation process follows a pseudo-first-order reaction which is accompanied by irreversible aggregation and intramolecular self-crosslinking of the enzyme. Although the mutations are mostly located on the surface of the enzyme, kinetic constants determined with the standard substrate CBZ-Gln-Gly-OH revealed a decrease in KM from 8.6 mM (± 0.1) to 3.5 mM (± 0.1) for the recombinant wild-type MTG and TG16, respectively. The improved performance of TG16 at higher temperatures is exemplary demonstrated with the crosslinking of the substrate protein β-casein at 60 °C. Using molecular dynamics simulations, it was shown that the increased thermoresistance is caused by a higher backbone rigidity as well as increased hydrophobic interactions and newly formed hydrogen bridges.
Publikation
Nothias, L.-F.; Petras, D.; Schmid, R.; Dührkop, K.; Rainer, J.; Sarvepalli, A.; Protsyuk, I.; Ernst, M.; Tsugawa, H.; Fleischauer, M.; Aicheler, F.; Aksenov, A. A.; Alka, O.; Allard, P.-M.; Barsch, A.; Cachet, X.; Caraballo-Rodriguez, A. M.; Da Silva, R. R.; Dang, T.; Garg, N.; Gauglitz, J. M.; Gurevich, A.; Isaac, G.; Jarmusch, A. K.; Kameník, Z.; Kang, K. B.; Kessler, N.; Koester, I.; Korf, A.; Le Gouellec, A.; Ludwig, M.; Martin H., C.; McCall, L.-I.; McSayles, J.; Meyer, S. W.; Mohimani, H.; Morsy, M.; Moyne, O.; Neumann, S.; Neuweger, H.; Nguyen, N. H.; Nothias-Esposito, M.; Paolini, J.; Phelan, V. V.; Pluskal, T.; Quinn, R. A.; Rogers, S.; Shrestha, B.; Tripathi, A.; van der Hooft, J. J. J.; Vargas, F.; Weldon, K. C.; Witting, M.; Yang, H.; Zhang, Z.; Zubeil, F.; Kohlbacher, O.; Böcker, S.; Alexandrov, T.; Bandeira, N.; Wang, M.; Dorrestein, P. C.;Feature-based molecular networking in the GNPS analysis environmentNature Methods17905-908(2020)DOI: 10.1038/s41592-020-0933-6
Molecular networking has become a key method to visualize and annotate the chemical space in non-targeted mass spectrometry data. We present feature-based molecular networking (FBMN) as an analysis method in the Global Natural Products Social Molecular Networking (GNPS) infrastructure that builds on chromatographic feature detection and alignment tools. FBMN enables quantitative analysis and resolution of isomers, including from ion mobility spectrometry.