Fig. 2: The consensus mechanism of wild type and engineered AOS enzymes.
The enzyme resting state is that of an “internal aldimine” or Schiff base, in which the PLP cofactor is covalently bound to the enzyme via a conserved Lys residue (Pi = phosphate). The first chemical step is reversible transimination of the internal aldimine by an amino-acid to generate a PLP:amino acid “external aldimine”. In the wild type AOS a conformational change is caused by binding of the acyl-CoA substrate that rotates the external aldimine into the “Dunathan conformation”. This permits the catalytic lysine to deprotonate the amino-acid at Cα and generate a reactive PLP:quinonoid intermediate (observed at 490-510 nm). The nucleophilic quinonoid reacts with the electrophilic acyl-CoA thioester in a Claisen-like condensation to form the C-C bond of a β-ketoacid intermediate and eliminates CoASH irreversibly (detected by the DTNB assay). The β-ketoacid is decarboxylated to generate a PLP:α-aminoketone product external aldimine, which finally reacts with the Lys residue to release the α-aminoketone product and return the AOS to the internal aldimine. In blue the engineered ThAOS V79 variants produced in this study have been shown to generate the PLP:quinonoid in the absence of the acyl-CoA substrate. This expands the substrate scope to allow binding and reaction between a range of amino acids, acyl-CoAs and acyl-SNAc substrates.
