Phenylalanine Ammonia‐Lyase‐Catalyzed Deamination of an Acyclic Amino Acid: Enzyme Mechanistic Studies Aided by a Novel Microreactor Filled with Magnetic Nanoparticles |
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| Authors: | Diána Weiser Dr. László Csaba Bencze Gergely Bánóczi Ferenc Ender Dr. Róbert Kiss Eszter Kókai Dr. András Szilágyi Prof. Dr. Beáta G. Vértessy Dr. Ödön Farkas Dr. Csaba Paizs Prof. Dr. László Poppe |
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| Affiliation: | 1. Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Budapest, Hungary;2. Babe?-Bolyai University of Cluj-Napoca, Cluj-Napoca, Romania;3. Department of Electron Devices, Budapest University of Technology and Economics, Budapest, Hungary;4. Gedeon Richter Plc. Gy?mr?i út 19-21, Budapest, Hungary;5. Department of Physical Chemistry and Materials Science, Budapest University of Technology and Economics, Budapest, Hungary;6. Institute of Enzymology, Research Centre for Natural Sciences of Hungarian Academy of Sciences, Budapest, Hungary;7. Department of Biotechnology and Food Sciences, Budapest University of Technology and Economics, Budapest, Hungary;8. Department of Organic Chemistry, E?tv?s Lóránd University, Budapest, Hungary;9. SynBiocat Ltd., Budapest, Hungary |
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| Abstract: | Phenylalanine ammonia‐lyase (PAL), found in many organisms, catalyzes the deamination of l ‐phenylalanine (Phe) to (E)‐cinnamate by the aid of its MIO prosthetic group. By using PAL immobilized on magnetic nanoparticles and fixed in a microfluidic reactor with an in‐line UV detector, we demonstrated that PAL can catalyze ammonia elimination from the acyclic propargylglycine (PG) to yield (E)‐pent‐2‐ene‐4‐ynoate. This highlights new opportunities to extend MIO enzymes towards acyclic substrates. As PG is acyclic, its deamination cannot involve a Friedel–Crafts‐type attack at an aromatic ring. The reversibility of the PAL reaction, demonstrated by the ammonia addition to (E)‐pent‐2‐ene‐4‐ynoate yielding enantiopure l ‐PG, contradicts the proposed highly exothermic single‐step mechanism. Computations with the QM/MM models of the N‐MIO intermediates from l ‐PG and l ‐Phe in PAL show similar arrangements within the active site, thus supporting a mechanism via the N‐MIO intermediate. |
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| Keywords: | enzyme catalysis magnetic nanoparticles microreactors phenylalanine ammonia lyase reaction mechanisms |
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