6DVS

Crystal structure of Pseudomonas stutzeri D-phenylglycine aminotransferase

6DVS の概要

• エントリーDOI: 10.2210/pdb6dvs/pdb
• 分子名称: D-phenylglycine aminotransferase, SULFATE ION, 1,2-ETHANEDIOL, ... (7 entities in total)
• 機能のキーワード: aminotransferase, transferase
• 由来する生物種: Pseudomonas stutzeri (Pseudomonas perfectomarina)
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 50639.76

構造登録者

Couture, J.F.,Chica, R. (登録日: 2018-06-25, 公開日: 2018-09-12, 最終更新日: 2024-03-13)

主引用文献

Walton, C.J.W.,Thiebaut, F.,Brunzelle, J.S.,Couture, J.F.,Chica, R.A.
Structural Determinants of the Stereoinverting Activity of Pseudomonas stutzeri d-Phenylglycine Aminotransferase.
Biochemistry, 57:5437-5446, 2018

PubMed 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 great potential as a biocatalyst for the synthesis of d-amino acids is the stereoinverting 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.

PubMed: 30153007
DOI: 10.1021/acs.biochem.8b00767

実験手法

X-RAY DIFFRACTION (1.821 Å)