基于结构工程的最小脯氨酸脱氢酶结构域抑制剂发现。

IF 2.6 4区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Protein Engineering Design & Selection Pub Date : 2022-02-17 DOI:10.1093/protein/gzac016
Alexandra N Bogner, Juan Ji, John J Tanner
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引用次数: 0

摘要

脯氨酸脱氢酶(PRODH)可以催化fad依赖的l-脯氨酸氧化生成Δ1-pyrroline-5-carboxylate,由于其在癌细胞代谢中的重要性,因此是发现抑制剂的靶标。由于人类PRODH难以纯化,细菌双功能酶脯氨酸利用A (PutA)的PRODH结构域已被用于抑制剂的开发。由于多肽链长、构象灵活性和与PRODH活性无关的结构域的存在,这些系统具有局限性。在此,我们报道了用于抑制剂发现的最小PRODH结构域的工程。最好的设计包含了来自Sinorhizobium meliloti的1233个PutA残基的三分之一,并包含了一个取代PutA α-结构域的连接体。最小的PRODHs表现出接近野生型的酶活性,并且对已知的抑制剂和灭活剂敏感。在1.23和1.72 Å分辨率下测定了S-(-)-四氢-2-呋喃酸和2-(呋喃-2-基)乙酸抑制的最小PRODHs的晶体结构。最小PRODHs在化学探针发现中应该是有用的。
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Structure-based engineering of minimal proline dehydrogenase domains for inhibitor discovery.

Proline dehydrogenase (PRODH) catalyzes the FAD-dependent oxidation of l-proline to Δ1-pyrroline-5-carboxylate and is a target for inhibitor discovery because of its importance in cancer cell metabolism. Because human PRODH is challenging to purify, the PRODH domains of the bacterial bifunctional enzyme proline utilization A (PutA) have been used for inhibitor development. These systems have limitations due to large polypeptide chain length, conformational flexibility and the presence of domains unrelated to PRODH activity. Herein, we report the engineering of minimal PRODH domains for inhibitor discovery. The best designs contain one-third of the 1233-residue PutA from Sinorhizobium meliloti and include a linker that replaces the PutA α-domain. The minimal PRODHs exhibit near wild-type enzymatic activity and are susceptible to known inhibitors and inactivators. Crystal structures of minimal PRODHs inhibited by S-(-)-tetrahydro-2-furoic acid and 2-(furan-2-yl)acetic acid were determined at 1.23 and 1.72 Å resolution. Minimal PRODHs should be useful in chemical probe discovery.

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来源期刊
Protein Engineering Design & Selection
Protein Engineering Design & Selection 生物-生化与分子生物学
CiteScore
3.30
自引率
4.20%
发文量
14
审稿时长
6-12 weeks
期刊介绍: Protein Engineering, Design and Selection (PEDS) publishes high-quality research papers and review articles relevant to the engineering, design and selection of proteins for use in biotechnology and therapy, and for understanding the fundamental link between protein sequence, structure, dynamics, function, and evolution.
期刊最新文献
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