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.
Highlights
In nature, enzymes with phenylalanine ammonia lyase (PAL) activity selectively bind phenylalanine (S)-3a and catalyse its deamination, forming cinnamate 1a and ammonia [1, 2]
Is located on an active site loop lid [6]. It has been shown through thermal activity studies of (S)-selective phenylalanine aminomutase (PAM) [7] and mutagenesis with an (R)-β-forming aminomutase [8] that the dynamics and flexibility of this inner active site loop dictate whether an overall lyase or mutase reaction occurs
This is mediated in part by positioning of the essential tyrosine in an optimal position for enzyme catalysis, with a stable, long-lived closed conformation increasing the likelihood of reamination occurring before loop movements mediating release of product(s) from the active site [7,8,9]
Summary
Enzymes with phenylalanine ammonia lyase (PAL) activity selectively bind phenylalanine (S)-3a and catalyse its deamination, forming cinnamate 1a and ammonia [1, 2]. It has been shown through thermal activity studies of (S)-selective PAMs [7] and mutagenesis with an (R)-β-forming aminomutase [8] that the dynamics and flexibility of this inner active site loop dictate whether an overall lyase or mutase reaction occurs. We report the discovery of significant levels of β-amino acid formation with certain substrates using an extensively studied PAL biocatalyst with no previously identified aminomutase activity.
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