α-Oxoamine synthases for industrial biocatalysis
Item statusRestricted Access
Embargo end date18/04/2023
α-Oxoamine synthase (AOS) enzymes are key pyridoxal-5’-phosphate (PLP)-dependent, C-C bond-forming enzymes, which catalyse a Claisen-like condensation reaction between an L-α-amino-acid (L-AA) and an acyl-CoA thioester, with loss of CO2 and CoASH, to yield chiral α-aminoketones. AOS enzymes are the first and rate-limiting biosynthetic steps in the biosyntheses of many important metabolites, including heme, biotin and sphingolipids. By virtue of the release of CoASH and CO2, AOS enzymes are inherently irreversible. Since the publication of the first enzyme example in 1958, their reactivity, mechanism, structure and biological relevance have been studied extensively, with new papers being published every year continuously for the past six decades. Recent years have seen a rapid acceleration in the rate of discovery and publication of new AOS enzymes, greatly expanding the number both of enzyme analogues and substrates. It is only very recently however that AOSs have been considered as useful biocatalysts. This is primarily due to 2 key challenges; the oftentimes strong substrate specificity of AOSs for the AA and thioester substrates; and the inherent requirement of stoichiometric amounts of expensive CoA-thioester as the electrophile. In this report, the full catalytic cycle of AOS enzymes is exploited for the first time. A thermophilic and moderately substrate-promiscuous AOS from the literature, ThAOS from Thermus thermophilus, is recombinantly expressed and purified from E. coli, and characterised as a potentially useful C-C bond-forming biocatalyst. Steady-state kinetic and UV-vis-based spectroscopic assays are used to probe the enzyme’s reactivity, successfully showing it to exhibit a broader substrate scope than previously thought. ThAOS was shown to catalyse 20 Claisen-like condensations between four unique AA substrates and five different acyl-CoA thioesters. The ThAOS Claisen-condensation step was next successfully coupled in situ with a chemical step, the Knorr pyrrole reaction (KPR) at elevated temperatures to generate Knorr pyrroles in excellent analytical yields in as little as 2 hours. Substrate tolerance is also probed for the KPR acceptor reagent, illustrating the full scope of pyrroles accessible via our chemo-biocatalytic cascade and demonstrating the formation of >25 pyrrole products. Lastly, the economics of the cascade are optimised by the obtention and testing of a variety of auxiliary biocatalysts to improve the yield reduce the wastage of the cascade. The crystal structure of the ThAOS biocatalyst is also reported to 1.6 Å resolution, paving the way for future mutagenesis and engineering. Having shown the usefulness of ThAOS as a C-C bond-forming biocatalyst and obtained its crystal structure, the enzyme was next evolved to improve its activity and substrate scope. Inspection of the high-resolution crystal structure of ThAOS revealed an active-site loop which protrudes into the substrate-binding pocket. Mutagenesis of two key residues on this loop yielded mutants which greatly improved upon the wild-type (WT) ThAOS enzyme in all dimensions tested. A total of four mutants were obtained with substrate scope beyond that of WT ThAOS, significantly expanding the number and nature of α-aminoketones obtainable using the enzyme. Mutants were also obtained which were up to 65× more efficient than the WT enzyme, and with improved thermostability, able to withstand incubation at up to 90 °C. Lastly, the strong selectivity of the enzyme for acyl-CoA thioesters was significantly diminished, the mutants being able to turn over cheaper and more stable substrate analogues. This work establishes ThAOS as a potent C-C bond-forming biocatalyst, which with further engineering and evolution could reach the level of being useful in an industrial or synthetic context.