Structural and Mechanistic Analysis of Drosophila melanogaster Agmatine N-Acetyltransferase, an Enzyme that Catalyzes the Formation of N-Acetylagmatine
Abstract Agmatine N-acetyltransferase (AgmNAT) catalyzes the formation of N-acetylagmatine from acetyl-CoA and agmatine. Herein, we provide evidence that Drosophila melanogaster AgmNAT (CG15766) catalyzes the formation of N-acetylagmatine using an ordered sequential mechanism; acetyl-CoA binds prior...
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2017
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oai:doaj.org-article:b2083b0e3aaf4cd49387f1ef030550cb2021-12-02T15:05:00ZStructural and Mechanistic Analysis of Drosophila melanogaster Agmatine N-Acetyltransferase, an Enzyme that Catalyzes the Formation of N-Acetylagmatine10.1038/s41598-017-13669-62045-2322https://doaj.org/article/b2083b0e3aaf4cd49387f1ef030550cb2017-10-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-13669-6https://doaj.org/toc/2045-2322Abstract Agmatine N-acetyltransferase (AgmNAT) catalyzes the formation of N-acetylagmatine from acetyl-CoA and agmatine. Herein, we provide evidence that Drosophila melanogaster AgmNAT (CG15766) catalyzes the formation of N-acetylagmatine using an ordered sequential mechanism; acetyl-CoA binds prior to agmatine to generate an AgmNAT•acetyl-CoA•agmatine ternary complex prior to catalysis. Additionally, we solved a crystal structure for the apo form of AgmNAT with an atomic resolution of 2.3 Å, which points towards specific amino acids that may function in catalysis or active site formation. Using the crystal structure, primary sequence alignment, pH-activity profiles, and site-directed mutagenesis, we evaluated a series of active site amino acids in order to assign their functional roles in AgmNAT. More specifically, pH-activity profiles identified at least one catalytically important, ionizable group with an apparent pKa of ~7.5, which corresponds to the general base in catalysis, Glu-34. Moreover, these data led to a proposed chemical mechanism, which is consistent with the structure and our biochemical analysis of AgmNAT.Daniel R. DempseyDerek A. NicholsMatthew R. BattistiniOrville PembertonSantiago Rodriguez OspinaXiujun ZhangAnne-Marie CarpenterBrian G. O’FlynnJames W. LeahyAnkush KanwarEric M. LewandowskiYu ChenDavid J. MerklerNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-15 (2017) |
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Medicine R Science Q |
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Medicine R Science Q Daniel R. Dempsey Derek A. Nichols Matthew R. Battistini Orville Pemberton Santiago Rodriguez Ospina Xiujun Zhang Anne-Marie Carpenter Brian G. O’Flynn James W. Leahy Ankush Kanwar Eric M. Lewandowski Yu Chen David J. Merkler Structural and Mechanistic Analysis of Drosophila melanogaster Agmatine N-Acetyltransferase, an Enzyme that Catalyzes the Formation of N-Acetylagmatine |
| description |
Abstract Agmatine N-acetyltransferase (AgmNAT) catalyzes the formation of N-acetylagmatine from acetyl-CoA and agmatine. Herein, we provide evidence that Drosophila melanogaster AgmNAT (CG15766) catalyzes the formation of N-acetylagmatine using an ordered sequential mechanism; acetyl-CoA binds prior to agmatine to generate an AgmNAT•acetyl-CoA•agmatine ternary complex prior to catalysis. Additionally, we solved a crystal structure for the apo form of AgmNAT with an atomic resolution of 2.3 Å, which points towards specific amino acids that may function in catalysis or active site formation. Using the crystal structure, primary sequence alignment, pH-activity profiles, and site-directed mutagenesis, we evaluated a series of active site amino acids in order to assign their functional roles in AgmNAT. More specifically, pH-activity profiles identified at least one catalytically important, ionizable group with an apparent pKa of ~7.5, which corresponds to the general base in catalysis, Glu-34. Moreover, these data led to a proposed chemical mechanism, which is consistent with the structure and our biochemical analysis of AgmNAT. |
| format |
article |
| author |
Daniel R. Dempsey Derek A. Nichols Matthew R. Battistini Orville Pemberton Santiago Rodriguez Ospina Xiujun Zhang Anne-Marie Carpenter Brian G. O’Flynn James W. Leahy Ankush Kanwar Eric M. Lewandowski Yu Chen David J. Merkler |
| author_facet |
Daniel R. Dempsey Derek A. Nichols Matthew R. Battistini Orville Pemberton Santiago Rodriguez Ospina Xiujun Zhang Anne-Marie Carpenter Brian G. O’Flynn James W. Leahy Ankush Kanwar Eric M. Lewandowski Yu Chen David J. Merkler |
| author_sort |
Daniel R. Dempsey |
| title |
Structural and Mechanistic Analysis of Drosophila melanogaster Agmatine N-Acetyltransferase, an Enzyme that Catalyzes the Formation of N-Acetylagmatine |
| title_short |
Structural and Mechanistic Analysis of Drosophila melanogaster Agmatine N-Acetyltransferase, an Enzyme that Catalyzes the Formation of N-Acetylagmatine |
| title_full |
Structural and Mechanistic Analysis of Drosophila melanogaster Agmatine N-Acetyltransferase, an Enzyme that Catalyzes the Formation of N-Acetylagmatine |
| title_fullStr |
Structural and Mechanistic Analysis of Drosophila melanogaster Agmatine N-Acetyltransferase, an Enzyme that Catalyzes the Formation of N-Acetylagmatine |
| title_full_unstemmed |
Structural and Mechanistic Analysis of Drosophila melanogaster Agmatine N-Acetyltransferase, an Enzyme that Catalyzes the Formation of N-Acetylagmatine |
| title_sort |
structural and mechanistic analysis of drosophila melanogaster agmatine n-acetyltransferase, an enzyme that catalyzes the formation of n-acetylagmatine |
| publisher |
Nature Portfolio |
| publishDate |
2017 |
| url |
https://doaj.org/article/b2083b0e3aaf4cd49387f1ef030550cb |
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