Phenylalanine Ammonia‐Lyase‐Catalyzed Deamination of an Acyclic Amino Acid: Enzyme Mechanistic Studies Aided by a Novel Microreactor Filled with Magnetic Nanoparticles

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.

Tailored Mutants of Phenylalanine Ammonia‐Lyase from Petroselinum crispum for the Synthesis of Bulky l‐ and d‐Arylalanines

“…As future perspectives, the combination of tailored Pc PAL mutants with the immobilization techniques reported recently can lead to their use in continuous‐flow reactors, which provides accessibility for the industrial synthesis of sterically demanding non‐natural arylalanines.…”

Pseudomonas fluorescensStrain R124 Encodes Three Different MIO Enzymes

“…PALs exhibit high similarity to TALs and PTALs and accept a broad range of arylalanines as substrates, such as various substituted phenylalanines, heteroarylalanines, and even propargylglycine . A single amino acid in the aromatic binding region of the active site was identified as a critical position switching between PAL and TAL activities in several cases.…”

Geometric optimization of microreactor chambers to increase the homogeneity of the velocity field

“…The accumulated particles form a thin biocatalyst layer in the microchambers while the magnet placed beneath the chamber keeps the particles in the chamber volume despite the liquid flow through the chambers (figure 1(c)). The inhomogeneous phase packed-bed microreactor formed in this way creates a unique opportunity to develop modular microsystems with the ability to allow flexible variation of the biocatalysts [2]. Performing enzymatic reactions in such microreactors has many advantages due to their reduced (Some figures may appear in colour only in the online journal)…”