半胱氨酸翻译后修饰gcn5相关n -乙酰转移酶的结构、功能和调控评价

IF 2.5 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Biochemical and biophysical research communications Pub Date : 2025-01-11 DOI:10.1016/j.bbrc.2025.151299
Patricia Uychoco , Karolina A. Majorek , Ashley N. Ives , Van Thi Bich Le , Pamela L. Caro De Silva , Vanessa L. Paurus , Isaac Kwame Attah , Mary S. Lipton , Wladek Minor , Misty L. Kuhn
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引用次数: 0

摘要

细胞内的多胺受到精胺/精胺n -乙酰转移酶(SSAT)的严格调控。虽然已经在不同的细菌物种中研究了几种ssat,但在许多生物体中,关于哪些蛋白质是功能性ssat的知识仍然存在重大差距。例如,虽然已知铜绿假单胞菌合成多胺亚精胺,但使该分子乙酰化的SSAT及其在调节细胞内多胺中的重要性尚不清楚。我们之前从P. aeruginosa (PA2271)中发现了一个候选gcn5相关的n -乙酰转移酶(GNAT)蛋白,该蛋白可以使亚精胺乙酰化,因此可以发挥这一作用,但没有进一步的研究。在这里,我们通过测定PA2271蛋白的x射线晶体结构和进行酶动力学分析来探索其结构/功能关系。我们还确定了催化和底物结合所必需的活性位点残基。随着研究的进展,我们遇到的结果促使我们探索四种半胱氨酸残基对酶活性和二硫键形成或半胱氨酸残基修饰的重要性。我们发现PA2271中的这些半胱氨酸残基对蛋白质的溶解度和活性很重要,并且在这些影响中存在半胱氨酸残基之间的相互关系。此外,我们还发现C121和C165之间可以形成二硫键,推测这些残基可能参与了PA2271蛋白活性的氧化还原调节。
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Structural, functional, and regulatory evaluation of a cysteine post-translationally modified Gcn5-related N-acetyltransferase
Polyamines within the cell are tightly regulated by spermidine/spermine N-acetyltransferase (SSAT) enzymes. While several SSATs have been investigated in different bacterial species, there is still a significant gap in knowledge about which proteins are functional SSATs in many organisms. For example, while it is known that Pseudomonas aeruginosa synthesizes the polyamine spermidine, the SSAT that acetylates this molecule and its importance in regulating intracellular polyamines remains unknown. We previously identified a candidate Gcn5-related N-acetyltransferase (GNAT) protein from P. aeruginosa (PA2271) that could fulfill this role since it acetylates spermidine, but no further studies were conducted. Here, we explored the structure/function relationship of the PA2271 protein by determining its X-ray crystal structure and performing enzyme kinetics assays. We also identified active site residues that are essential for catalysis and substrate binding. As the study progressed, we encountered results that led us to explore the importance of four cysteine residues on enzyme activity and disulfide bond formation or modification of cysteine residues. We found these cysteine residues in PA2271 are important for protein solubility and activity, and there is an interrelationship between cysteine residues that contribute to these effects. Furthermore, we also found disulfide bonds could form between C121 and C165 and speculate that these residues may contribute to redox regulation of PA2271 protein activity.
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来源期刊
Biochemical and biophysical research communications
Biochemical and biophysical research communications 生物-生化与分子生物学
CiteScore
6.10
自引率
0.00%
发文量
1400
审稿时长
14 days
期刊介绍: Biochemical and Biophysical Research Communications is the premier international journal devoted to the very rapid dissemination of timely and significant experimental results in diverse fields of biological research. The development of the "Breakthroughs and Views" section brings the minireview format to the journal, and issues often contain collections of special interest manuscripts. BBRC is published weekly (52 issues/year).Research Areas now include: Biochemistry; biophysics; cell biology; developmental biology; immunology ; molecular biology; neurobiology; plant biology and proteomics
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