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
{"title":"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","authors":"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","doi":"10.1016/j.bbrc.2025.151299","DOIUrl":null,"url":null,"abstract":"<div><div>Polyamines within the cell are tightly regulated by spermidine/spermine <em>N</em>-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 <em>Pseudomonas aeruginosa</em> 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 <em>N-</em>acetyltransferase (GNAT) protein from <em>P. aeruginosa</em> (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<sub>121</sub> and C<sub>165</sub> and speculate that these residues may contribute to redox regulation of PA2271 protein activity.</div></div>","PeriodicalId":8779,"journal":{"name":"Biochemical and biophysical research communications","volume":"748 ","pages":"Article 151299"},"PeriodicalIF":2.5000,"publicationDate":"2025-01-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Structural, functional, and regulatory evaluation of a cysteine post-translationally modified Gcn5-related N-acetyltransferase\",\"authors\":\"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\",\"doi\":\"10.1016/j.bbrc.2025.151299\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><div>Polyamines within the cell are tightly regulated by spermidine/spermine <em>N</em>-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 <em>Pseudomonas aeruginosa</em> 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 <em>N-</em>acetyltransferase (GNAT) protein from <em>P. aeruginosa</em> (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<sub>121</sub> and C<sub>165</sub> and speculate that these residues may contribute to redox regulation of PA2271 protein activity.</div></div>\",\"PeriodicalId\":8779,\"journal\":{\"name\":\"Biochemical and biophysical research communications\",\"volume\":\"748 \",\"pages\":\"Article 151299\"},\"PeriodicalIF\":2.5000,\"publicationDate\":\"2025-01-11\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Biochemical and biophysical research communications\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0006291X25000130\",\"RegionNum\":3,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biochemical and biophysical research communications","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0006291X25000130","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
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.
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