Phenylalanine Ammonia-Lyase-Catalyzed Deamination of an Acyclic Amino Acid: Enzyme Mechanistic Studies Aided by a Novel Microreactor Filled with Magnetic Nanoparticles.
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.
Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Műegyetem rkp. 3, 1111, Budapest, Hungary.
Diána
D
Weiser
Babeş-Bolyai University of Cluj-Napoca, Arany János str. 11, 400028, Cluj-Napoca, Romania.
László Csaba
LC
Bencze
Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Műegyetem rkp. 3, 1111, Budapest, Hungary.
Gergely
G
Bánóczi
Department of Electron Devices, Budapest University of Technology and Economics, Magyar tudósok körútja 2, 1117, Budapest, Hungary.
Ferenc
F
Ender
Gedeon Richter Plc. Gyömrői út 19-21, 1103, Budapest, Hungary.
Róbert
R
Kiss
Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Műegyetem rkp. 3, 1111, Budapest, Hungary.
Eszter
E
Kókai
Department of Physical Chemistry and Materials Science, Budapest University of Technology and Economics, Budafoki út 8, 1111, Budapest, Hungary.
András
A
Szilágyi
Institute of Enzymology, Research Centre for Natural Sciences of Hungarian Academy of Sciences, Magyar tudósok körútja 2, 1117, Budapest, Hungary.|Department of Biotechnology and Food Sciences, Budapest University of Technology and Economics, Szt. Gellért tér 4, 1111, Budapest, Hungary.
Beáta G
BG
Vértessy
Department of Organic Chemistry, Eötvös Lóránd University, Pázmány Péter sétány 1A, 1117, Budapest, Hungary.
Ödön
Ö
Farkas
Babeş-Bolyai University of Cluj-Napoca, Arany János str. 11, 400028, Cluj-Napoca, Romania. paizs@chem.ubbcluj.ro.
Csaba
C
Paizs
Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Műegyetem rkp. 3, 1111, Budapest, Hungary. poppe@mail.bme.hu.|SynBiocat Ltd., Lázár deák u 4/1, 1173, Budapest, Hungary. poppe@mail.bme.hu.
László
L
Poppe
D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D003641:Deamination; D007700:Kinetics; D058185:Magnetite Nanoparticles / Q000737:chemistry*; D046210:Microfluidic Analytical Techniques; D008958:Models, Molecular; D010650:Phenylalanine Ammonia-Lyase / Q000737:chemistry / Q000378:metabolism*; D011789:Quantum Theory
D000596:Amino Acids; D058185:Magnetite Nanoparticles; D010650:Phenylalanine Ammonia-Lyase
enzyme catalysis; magnetic nanoparticles; microreactors; phenylalanine ammonia lyase; reaction mechanisms