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Issue 19, 2020
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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

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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 α-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.

Graphical abstract: d-Phenylglycine aminotransferase (d-PhgAT) – substrate scope and structural insights of a stereo-inverting biocatalyst used in the preparation of aromatic amino acids

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Article information


Submitted
10 Jul 2020
Accepted
27 Jul 2020
First published
28 Jul 2020

This article is Open Access

Catal. Sci. Technol., 2020,10, 6533-6543
Article type
Paper

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

A. Serpico, S. De Cesare, J. Marles-Wright, M. K. Akhtar, G. J. Loake and D. J. Campopiano, Catal. Sci. Technol., 2020, 10, 6533 DOI: 10.1039/D0CY01391A

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