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D-phenylglycine aminotransferase (D-PhgAT) – substrate scope and structural insights of a stereo-inverting biocatalyst used in the preparation of aromatic amino acids

DOI: 10.1039/D0CY01391A DOI Help

Authors: Annabel Serpico (University of Edinburgh; LEITAT Technological Center) , Silvia De Cesare (University of Edinburgh) , Jon Marles-wright (Newcastle University) , M. Kalim Akhtar (University of Edinburgh; United Arab Emirates University) , Gary Loake (University of Edinburgh) , Dominic James Campopiano (University of Edinburgh)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Catalysis Science & Technology

State: Published (Approved)
Published: July 2020

Open Access Open Access

Abstract: Enantiopure amines are key building blocks in the synthesis of many pharmaceuticals, so a route to their production is a current goal for biocatalysis. The stereo-inverting D-phenylglycine aminotransferase (D-PhgAT), isolated from Pseudomonas stutzeri ST-201, catalyses the reversible transamination from L-glutamic acid to benzoylformate, yielding -ketoglutarate and D-phenylglycine (D-Phg). Detailed kinetic analysis revealed a range of amine donor and acceptor substrates that allowed the synthesis of enantiopure aromatic D-amino acids at a preparative scale. We also determined the first X-ray crystal structure of D-PhgAT with its bound pyridoxal 5’-phosphate (PLP) cofactor at 2.25 Å resolution. A combination of structural analysis and site-directed mutagenesis of this class III aminotransferase revealed key residues that are potentially involved in the dual substrate recognition, as well as controlling the stereo-inverting behaviour of D-PhgAT. Two arginine residues (Arg34 and Arg407) are involved in substrate recognition within P and O binding pockets respectively. These studies lay the foundation for further enzyme engineering and promote D-PhgAT as a useful biocatalyst for the sustainable production of high value, aromatic D-amino acids.

Subject Areas: Chemistry

Instruments: I03-Macromolecular Crystallography


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