Discovery and Investigation of Mutase-like Activity in a Phenylalanine Ammonia Lyase from Anabaena variabilis

Abstract

The effect of extended reaction times on the regio-and enantioselectivity of the phenylalanine ammonia lyase (PAL)-catalysed amination of a subset of cinnamate derivatives was investigated. This was done using a PAL from the cyanobacterium Anabaena variabilis and incubation in a concentrated ammonia buffer. Whilst early time point analyses revealed excellent selectivities to give mostly the well-documented (S)-α-amino acid products, subsequent accumulation of other regio-/stereo-isomers was seen. For many para-substituted substrates, the β-regioisomer, a previously-unreported product with this enzyme class, was found to become more abundant than the α-, after sufficient incubation, with slight preference for the (R)-enantiomer. Although attempts to tune the selectivity of the PAL toward any of the three side products were largely unsuccessful, the results provide insight into the evolutionary history of this class of enzymes and reinforce the prominence of the toolbox of specific and selective cinnamate-aminating enzymes.

Engineered Ammonia Lyases for the Production of Challenging Electron-Rich l-Phenylalanines

“…Longer incubation times (48 h) afforded higher yields of the side product (Supporting Information, Table S1). The production of the β-regioisomer is presumably enhanced by the electronic properties of the substrates in this study, as seen with AvPAL and related aminomutases …”

“…In addition, with some of the variants we detected very small amounts of the corresponding β-amino acid as a side product. This is a side activity of PALs we have previously, 10 and that could be mitigated by incubating the biotransformation for shorter times (12−16 h). Longer incubation times (48 h) afforded higher yields of the side product (Supporting Information, Table S1).…”

Purification and Characterization of Anabaena flos-aquae Phenylalanine Ammonia-Lyase as a Novel Approach for Myristicin Biotransformation

“…The enzymatic yield of MMDA was 15.3 and 10.6% for PAL of S. platensis and A. flos-aquae using myristicin substrate, respectively. Similarly, PAL from A. variabilis [ 72 ] and Rhodotorula glutinis [ 20 ] transformed trans -cinnamic acid into L -phenylalanine with a yield of about 73% and 70%. In another work, Rhodotorula glutinis PAL produced L -phenylalanine methyl ester from trans -cinnamyl methyl ester in a biphasic system [ 73 ].…”

Recent Applications of Carbon‐Nitrogen Lyases in Asymmetric Synthesis of Noncanonical Amino Acids and Heterocyclic Compounds

“…Her current research interests include exploring the reactivity of enzymes from natural product biosynthesis pathways for producing pharmaceutically relevant molecules. [16][17][18][19][20][21] Ph, aryl H H RgPAL, AvPAL, PcPAL phenylalanine, β-aryl α-alanines [6,[22][23][24][25][26] chelating compound widely used in industry for a wide range of applications, such as soil remediation, paper manufacturing, waste water treatment and so on. (S,S)-EDDS was isolated as a secondary metabolite from the actinomycete Amycolatopsis japonicum, hypothetically serving as a scavenger of zinc (zincophore) and its bioproduction was strictly repressed by zinc.…”