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…
Seit Februar 2021 bietet Wolfgang Brandt, ehemaliger Leiter der Arbeitsgruppe Computerchemie am IPB, sein Citizen Science-Projekt zur Pilzbestimmung an. Dafür hat er in regelmäßigen Abständen öffentliche Vorträge zur Vielfalt…
Hashemi Haeri, H.; Schneegans, N.; Eisenschmidt-Bönn, D.; Brandt, W.; Wittstock, U.; Hinderberger, D.;Characterization of the active site in the thiocyanate-forming protein from Thlaspi arvense (TaTFP) using EPR spectroscopyBiol. Chem.405105-118(2024)DOI: 10.1515/hsz-2023-0187
Glucosinolates are plant thioglucosides, which act as chemical defenses. Upon tissue damage, their myrosinase-catalyzed hydrolysis yields aglucones that rearrange to toxic isothiocyanates. Specifier proteins such as thiocyanate-forming protein from Thlaspi arvense (TaTFP) are non-heme iron proteins, which capture the aglucone to form alternative products, e.g. nitriles or thiocyanates. To resolve the electronic state of the bound iron cofactor in TaTFP, we applied continuous wave electron paramagnetic resonance (CW EPR) spectroscopy at X-and Q-band frequencies (∼9.4 and ∼34 GHz). We found characteristic features of high spin and low spin states of a d5 electronic configuration and local rhombic symmetry during catalysis. We monitored the oxidation states of bound iron during conversion of allylglucosinolate by myrosinase and TaTFP in presence and absence of supplemented Fe2+. Without added Fe2+, most high spin features of bound Fe3+ were preserved, while different g’-values of the low spin part indicated slight rearrangements in the coordination sphere and/or structural geometry. We also examined involvement of the redox pair Fe3+/Fe2 in samples with supplemented Fe2+. The absence of any EPR signal related to Fe3+ or Fe2+ using an iron-binding deficient TaTFP variant allowed us to conclude that recorded EPR signals originated from the bound iron cofactor.
Publikation
Zheng, K.; Lyu, J. C.; Thomas, E. L.; Schuster, M.; Sanguankiattichai, N.; Ninck, S.; Kaschani, F.; Kaiser, M.; Hoorn, R. A.;The proteome of Nicotiana benthamiana is shaped by extensive protein processingNew Phytol.2431034-1049(2024)DOI: 10.1111/nph.19891
SummaryProcessing by proteases irreversibly regulates the fate of plant proteins and hampers the production of recombinant proteins in plants, yet only few processing events have been described in agroinfiltrated Nicotiana benthamiana, which has emerged as the main transient protein expression platform in plant science and molecular pharming.Here, we used in‐gel digests and mass spectrometry to monitor the migration and topography of 5040 plant proteins within a protein gel. By plotting the peptides over the gel slices, we generated peptographs that reveal where which part of each protein was detected within the protein gel.
These data uncovered that 60% of the detected proteins have proteoforms that migrate at lower than predicted molecular weights, implicating extensive proteolytic processing. This analysis confirms the proteolytic removal and degradation of autoinhibitory prodomains of most but not all proteases, and revealed differential processing within pectinemethylesterase and lipase families. This analysis also uncovered intricate processing of glycosidases and uncovered that ectodomain shedding might be common for a diverse range of receptor‐like kinases. Transient expression of double‐tagged candidate proteins confirmed processing events in vivo.
This large proteomic dataset implicates an elaborate proteolytic machinery shaping the proteome of N. benthamiana.
Publikation
Münch, J.; Dietz, N.; Barber-Zucker, S.; Seifert, F.; Matschi, S.; Püllmann, P.; Fleishman, S. J.; Weissenborn, M. J.;Functionally diverse peroxygenases by AlphaFold2, design, and signal peptide shufflingACS Catal.144738-4748(2024)DOI: 10.1021/acscatal.4c00883
Unspecific peroxygenases (UPOs) are fungal enzymes that attract significant attention for their ability to perform versatile oxyfunctionalization reactions using H2O2. Unlike other oxygenases, UPOs do not require additional reductive equivalents or electron transfer chains that complicate basic and applied research. Nevertheless, UPOs generally exhibit low to no heterologous production levels and only four UPO structures have been determined to date by crystallography limiting their usefulness and obstructing research. To overcome this bottleneck, we implemented a workflow that applies PROSS stability design to AlphaFold2 model structures of 10 unique and diverse UPOs followed by a signal peptide shuffling to enable heterologous production. Nine UPOs were functionally produced in Pichia pastoris, including the recalcitrant CciUPO and three UPOs derived from oomycetes the first nonfungal UPOs to be experimentally characterized. We conclude that the high accuracy and reliability of new modeling and design workflows dramatically expand the pool of enzymes for basic and applied research.
Publikation
Münch, J.; Soler, J.; Hünecke, N.; Homann, D.; Garcia-Borràs, M.; Weissenborn, M. J.;Computational-aided engineering of a selective unspecific peroxygenase toward enantiodivergent β-ionone hydroxylationACS Catal.138963-8972(2023)DOI: 10.1021/acscatal.3c00702
Unspecific peroxygenases (UPOs) perform oxy-functionalizations for a wide range of substrates utilizing H2O2 without the need for further reductive equivalents or electron transfer chains. Tailoring these promising enzymes toward industrial application was intensely pursued in the last decade with engineering campaigns addressing the heterologous expression, activity, stability, and improvements in chemo- and regioselectivity. One hitherto missing integral part was the targeted engineering of enantioselectivity for specific substrates with poor starting enantioselectivity. In this work, we present the engineering of the short-type MthUPO toward the enantiodivergent hydroxylation of the terpene model substrate, β-ionone. Guided by computational modeling, we designed a small smart library and screened it with a GC−MS setup. After two rounds of iterative protein evolution, the activity increased up to 17-fold and reached a regioselectivity of up to 99.6% for the 4-hydroxy-β-ionone. Enantiodivergent variants were identified with enantiomeric ratios of 96.6:3.4 (R) and 0.3:99.7 (S), respectively.