Fine‐tuning the activity and stability of an evolved enzyme active‐site through noncanonical amino‐acids

Abstract

Site-specific saturation mutagenesis within enzyme active sites can radically alter reaction specificity, though often with a trade-off in stability. Extending saturation mutagenesis with a range of noncanonical amino acids (ncAA) potentially increases the ability to improve activity and stability simultaneously. Previously, an Escherichia coli transketolase variant (S385Y/D469T/R520Q) was evolved to accept aromatic aldehydes not converted by wild-type. The aromatic residue Y385 was critical to the new acceptor substrate binding, and so was explored here beyond the natural aromatic residues, to probe side chain structure and electronics effects on enzyme function and stability. A series of five variants introduced decreasing aromatic ring electron density at position 385 in the order para-aminophenylalanine (pAMF), tyrosine (Y), phenylalanine (F), para-cyanophenylalanine (pCNF) and para-nitrophenylalanine (pNTF), and simultaneously modified the hydrogen-bonding potential of the aromatic substituent from accepting to donating. The fine-tuning of residue 385 yielded variants with a 43-fold increase in specific activity for 50 mM 3-HBA and 100% increased k cat (pCNF), 290% improvement in K m (pNTF), 240% improvement in k cat /K m (pAMF) and decreased substrate inhibition relative to Y. Structural modelling suggested switching of the ring-substituted functional group, from donating to accepting, stabilised a helix-turn (D259-H261) through an intersubunit H-bond with G262, to give a 7.8°C increase in the thermal transition mid-point, T m , and improved packing of pAMF. This is one of the first examples in which both catalytic activity and stability are simultaneously improved via site-specific ncAA incorporation into an enzyme active site, and further demonstrates the benefits of expanding designer libraries to include ncAAs.

Cellular and Biophysical Applications of Genetic Code Expansion

“…Likewise, Wilkinson and Dalby engineered mutations with transketolase, achieving increased activity and thermal stability by incorporating Phe analogues (Tyr and ncAAs 86, 102, and 108) with varied electron densities. 465 Among the amino acids, the ncAAs with a phenyl ring possessing higher electron density than tyrosine showed increased enzymatic activity. This increase of electron density enhanced π−π stacking interactions with the substrate, orienting it more favorably for catalysis and thus increasing the enzyme activity.…”

Engineering of enzymes using non-natural amino acids

“…Another example targeted the aromatic residue Phe385 in transketolase (TK), which has shown its critical role in new acceptor substrate binding. In 2020, this site was replaced with a series of derivatives of phenylalanine ( 1 – 3 ) on a previously created TK scaffold to reduce the aromatic ring electron density [ 30 ]. The results showed that the p -cyanophenylalanine ( p CNF, 3 ) mutant simultaneously increased the activity and stability of TK towards 3-hydroxybenzaldehyde (3-HBA), most likely due to the conversion of this functional group from an electron donor to an acceptor, which formed a new hydrogen bond with G262 to stabilise a local helix-turn structure.…”

“…To date, hundreds of nnAAs have been reported to be genetically encoded, but it is doubtful how many of these can be introduced by orthogonal translation systems at high efficiency and with high fidelity. In the research of our group, a methodology to quantify incorporation fidelity from deconvoluted mass spectra was introduced, and it was used to evaluate the incorporation performance of some of the most commonly used phenylalanine analogues in a specific site of TK [ 30 ]. Of these, p CNF had a misincorporation rate of around 22%, while it was as high as about 55% for p AMF.…”

An evolved pyrrolysyl-tRNA synthetase with polysubstrate specificity expands the toolbox for engineering enzymes with incorporation of noncanonical amino acids

“…Homoserine O -succinyltransferase from E. coli was engineered to have improved stability with randomly incorporated ncAAs achieved by the polyspecific aminoacyl-tRNA synthetase/tRNA pair (Li et al 2018 ). Also, three different ncAAs were incorporated into a transketolases variant from E. coli with the utility of p CNFRS to investigate the effect of aromatic ring electron density at a critical position on enzyme stability and catalysis efficiency (Wilkinson and Dalby 2021 ).…”