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Issue 19, 2020

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

Supplementary files

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

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

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