An ITPA Enzyme with Improved Substrate Selectivity

IF 1.9 4区生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY The Protein Journal Pub Date : 2023-12-08 DOI:10.1007/s10930-023-10162-0

Nicholas E. Burgis, Kandise VanWormer, Devin Robbins, Jonathan Smith

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Abstract

Recent clinical data have identified infant patients with lethal ITPA deficiencies. ITPA is known to modulate ITP concentrations in cells and has a critical function in neural development which is not understood. Polymorphism of the ITPA gene affects outcomes for both ribavirin and thiopurine based therapies and nearly one third of the human population is thought to harbor ITPA polymorphism. In a previous site-directed mutagenesis alanine screen of the ITPA substrate selectivity pocket, we identified the ITPA mutant, E22A, as a gain-of function mutant with enhanced ITP hydrolysis activity. Here we report a rational enzyme engineering experiment to investigate the biochemical properties of position 22 ITPA mutants and find that the E22D ITPA has two- and four-fold improved substrate selectivity for ITP over the canonical purine triphosphates ATP and GTP, respectively, while maintaining biological activity. The novel E22D ITPA should be considered as a platform for further development of ITPA therapies.

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一种具有更高底物选择性的 ITPA 酶

最近的临床数据发现，婴儿患者存在致命的 ITPA 缺乏症。据了解，ITPA 可调节细胞中的 ITP 浓度，在神经发育过程中起着至关重要的作用，但这一作用尚不清楚。ITPA 基因的多态性会影响利巴韦林和硫嘌呤类药物的治疗效果，近三分之一的人群被认为存在 ITPA 多态性。在之前对 ITPA 底物选择性口袋进行的定点突变丙氨酸筛选中，我们发现 ITPA 突变体 E22A 是一种功能增益突变体，具有增强的 ITP 水解活性。在这里，我们报告了一项合理的酶工程实验，以研究 ITPA 第 22 位突变体的生化特性，结果发现 E22D ITPA 对 ITP 的底物选择性分别比典型的嘌呤三磷酸 ATP 和 GTP 提高了 2 倍和 4 倍，同时还保持了生物活性。新型 E22D ITPA 应被视为进一步开发 ITPA 疗法的平台。

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来源期刊

The Protein Journal 生物-生化与分子生物学

CiteScore

5.20

自引率

0.00%

发文量

审稿时长

12 months

期刊介绍： The Protein Journal (formerly the Journal of Protein Chemistry) publishes original research work on all aspects of proteins and peptides. These include studies concerned with covalent or three-dimensional structure determination (X-ray, NMR, cryoEM, EPR/ESR, optical methods, etc.), computational aspects of protein structure and function, protein folding and misfolding, assembly, genetics, evolution, proteomics, molecular biology, protein engineering, protein nanotechnology, protein purification and analysis and peptide synthesis, as well as the elucidation and interpretation of the molecular bases of biological activities of proteins and peptides. We accept original research papers, reviews, mini-reviews, hypotheses, opinion papers, and letters to the editor.