Uychoco Patricia, Majorek Karolina A, Ives Ashley N, Le Van Thi Bich, Caro De Silva Pamela L, Paurus Vanessa L, Attah Isaac Kwame, Lipton Mary S, Minor Wladek, Kuhn Misty L
San Francisco State University, Department of Chemistry and Biochemistry, San Francisco, CA, USA.
University of Virginia, Department of Molecular Physiology and Biological Physics, Charlottesville, VA, USA.
Biochem Biophys Res Commun. 2025 Feb 8;748:151299. doi: 10.1016/j.bbrc.2025.151299. Epub 2025 Jan 11.
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 C and C and speculate that these residues may contribute to redox regulation of PA2271 protein activity.
细胞内的多胺由亚精胺/精胺N - 乙酰基转移酶（SSAT）严格调控。虽然已经在不同细菌物种中研究了几种SSAT，但对于许多生物体中哪些蛋白质是功能性SSAT，在认知上仍存在重大差距。例如，虽然已知铜绿假单胞菌能合成多胺亚精胺，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仍不清楚。我们之前从铜绿假单胞菌中鉴定出一种候选的Gcn5相关N - 乙酰基转移酶（GNAT）蛋白（PA2271），因其能使亚精胺乙酰化，可能发挥这一作用，但未进行进一步研究。在此，我们通过确定其X射线晶体结构并进行酶动力学分析，探索了PA2271蛋白的结构/功能关系。我们还鉴定了对催化和底物结合至关重要的活性位点残基。随着研究的推进，我们得到的结果促使我们探究四个半胱氨酸残基对酶活性以及半胱氨酸残基二硫键形成或修饰的重要性。我们发现PA2271中的这些半胱氨酸残基对蛋白质的溶解性和活性很重要，并且这些残基之间存在相互关系，共同促成这些效应。此外，我们还发现C与C之间可能形成二硫键，并推测这些残基可能有助于PA2271蛋白活性的氧化还原调节。
