Walton Curtis J W, Thiebaut Frédéric, Brunzelle Joseph S, Couture Jean-François, Chica Roberto A
Department of Chemistry and Biomolecular Sciences , University of Ottawa , Ottawa , Ontario , Canada K1N 6N5.
Centre for Catalysis Research and Innovation , University of Ottawa , Ottawa , Ontario , Canada K1N 6N5.
Biochemistry. 2018 Sep 18;57(37):5437-5446. doi: 10.1021/acs.biochem.8b00767. Epub 2018 Sep 7.
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.
芳香族 D-氨基酸是许多小分子治疗药物生产的关键前体。因此，开发其生物催化合成方法备受关注。一种有潜力作为 D-氨基酸合成生物催化剂的酶是来自施氏假单胞菌 ST-201 的立体转化 D-苯甘氨酸转氨酶（DPAT）。该酶催化一种独特的从 L 到 D 的转氨反应，通过一种尚不清楚的机制，从苯甲酰甲酸和 L-谷氨酸生成 D-苯甘氨酸和α-酮戊二酸。在这里，我们展示了 DPAT 的晶体结构，其表明该酶折叠成具有 III 类转氨酶代表性的双结构域结构。在晶体结构的指导下，我们进行了饱和诱变以探究该酶的底物结合口袋。这些实验帮助我们确定了两个精氨酸残基（R34 和 R407），每个结合口袋中各有一个，它们对催化作用至关重要。结合使用氨基酸底物库进行的动力学分析，我们的诱变和结构研究使我们能够提出一个解释 DPAT 双重 L/D 特异性的结合模型。我们的动力学分析还表明 DPAT 可以催化β-和γ-氨基酸的转氨反应，将这种酶重新归类为ω-转氨酶。总的来说，我们的研究强调 DPAT 活性位点适合进行蛋白质工程改造以扩大其底物范围，这为从廉价且丰富的 L-氨基酸合成各种有价值的光学纯 D-氨基酸生成新的生物催化剂提供了机会。
