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B6db references: 30153007

type Journal Article
authors Walton CJ, Thiebaut F, Brunzelle JS, Couture JF, Chica RA
title Structural determinants of the stereo-inverting activity of Pseudomonas stutzeri D-phenylglycine aminotransferase
journal Biochemistry
sel selected
ui 30153007
year (2018)
volume 57
number 37
pages 5437-5446
keywords doi: 10.1021/acs.biochem.8b00767
abstract Aromatic D-amino acids are key precursors for the production of many small molecule therapeutics. Therefore, the development of biocatalytic methods for their synthesis is of great interest. An enzyme that has high potential as a biocatalyst for the synthesis of D-amino acids is the stereo-inverting D-phenylglycine aminotransferase (DPAT) from Pseudomonas stutzeri ST-201. This enzyme catalyzes a unique L to D transamination reaction that produces D-phenylglycine and α-ketoglutarate from benzoylformate and L-glutamate, via a mechanism that is poorly understood. Here, we present the crystal structure of DPAT, which shows that the enzyme folds into a two-domain structure representative of class III aminotransferases. Guided by the crystal structure, we performed saturation mutagenesis to probe the substrate binding pockets of the enzyme. These experiments helped us identify two arginine residues (R34 and R407), one in each binding pocket, that are essential to catalysis. Together with kinetic analyses using a library of amino acid substrates, our mutagenesis and structural studies allow us to propose a binding model that explains the dual L/D specificity of DPAT. Our kinetic analyses also demonstrate that DPAT can catalyze the transamination of β- and γ-amino acids, reclassifying this enzyme as an ω-aminotransferase. Collectively, our studies highlight that the DPAT active site is amenable to protein engineering for expansion of its substrate scope, which offers the opportunity to generate new biocatalysts for the synthesis of a variety of valuable optically-pure D-amino acids from inexpensive and abundant L-amino acids.
last changed 2018/11/21 10:00

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