Structural, functional, and regulatory evaluation of a cysteine post-translationally modified Gcn5-related N-acetyltransferase

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.

doi:10.1016/j.bbrc.2025.151299

Title: Structural, functional, and regulatory evaluation of a cysteine post-translationally modified Gcn5-related N-acetyltransferase

Journal Article · 10 January 2025 · Biochemical and Biophysical Research Communications

DOI: https://doi.org/10.1016/j.bbrc.2025.151299 · OSTI ID:2506839

Uychoco, Patricia ^[1]; Majorek, Karolina A. ^[2]; Ives, Ashley N. ^[3];

^[1]; Caro De Silva, Pamela L. ^[1];

^[3];

^[3]; Minor, Wladek ^[2]; Kuhn, Misty L. ^[4]

San Francisco State Univ., CA (United States)
Univ. of Virginia, Charlottesville, VA (United States)
Pacific Northwest National Laboratory (PNNL), Richland, WA (United States). Environmental Molecular Sciences Laboratory (EMSL)
San Francisco State Univ., CA (United States); Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)

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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Research Organization:: Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)

Sponsoring Organization:: USDOE

Grant/Contract Number:: AC05-76RL01830

OSTI ID:: 2506839

Report Number(s):: PNNL-SA-207920

Journal Information:: Biochemical and Biophysical Research Communications, Journal Name: Biochemical and Biophysical Research Communications Vol. 748; ISSN 0006-291X

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
Pseudomonas aeruginosa
cysteine post-translational modifications
disulfide bonds
gcn5-related N-acetyltransferase (GNAT)
polyamine
spermidine

Title: Structural, functional, and regulatory evaluation of a cysteine post-translationally modified Gcn5-related N-acetyltransferase

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