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Updated: May 22, 2025

A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes
Published on: November 7, 2012
Rational engineering of a thermostable α-oxoamine synthase biocatalyst expands the substrate scope and synthetic
Ben Ashley1, Sam Mathew1, Mariyah Sajjad1
1School of Chemistry, University of Edinburgh, Joseph Black Building, David Brewster Road, Edinburgh, UK.
Abstract:
Carbon-carbon bond formation is one of the key pillars of organic synthesis. Green, selective and efficient biocatalytic methods for such are therefore highly desirable. The α-oxoamine synthases (AOSs) are a class of pyridoxal 5'-phosphate (PLP)-dependent, irreversible, carbon-carbon bond-forming enzymes, which have been limited previously by their narrow substrate specificity and requirement of acyl-CoA thioester substrates. We recently characterized a thermophilic enzyme from Thermus thermophilus (ThAOS) with a much broader substrate scope and described its use in a chemo-biocatalytic cascade process to generate pyrroles in good yields and timescales. Herein, we report the structure-guided engineering of ThAOS to arrive at variants able to use a greatly expanded range of amino acid and simplified N-acetylcysteamine (SNAc) acyl-thioester substrates. The crystal structure of the improved ThAOS V79A variant with a bound PLP:L-penicillamine external aldimine ligand, provides insight into the properties of the engineered biocatalyst.
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