2. Academic Validation
  3. Structural and Mechanistic Analysis of Drosophila melanogaster Agmatine N-Acetyltransferase, an Enzyme that Catalyzes the Formation of N-Acetylagmatine

Structural and Mechanistic Analysis of Drosophila melanogaster Agmatine N-Acetyltransferase, an Enzyme that Catalyzes the Formation of N-Acetylagmatine

  • Sci Rep. 2017 Oct 18;7(1):13432. doi: 10.1038/s41598-017-13669-6.
Daniel R Dempsey 1 2,  Derek A Nichols 3 4,  Matthew R Battistini 1,  Orville Pemberton 3,  Santiago Rodriguez Ospina 1,  Xiujun Zhang 3,  Anne-Marie Carpenter 1 5,  Brian G O'Flynn 1,  James W Leahy 1 3 6,  Ankush Kanwar 1,  Eric M Lewandowski 3,  Yu Chen 7,  David J Merkler 8
Affiliations

Affiliations

  • 1 Department of Chemistry, University of South Florida, Tampa, Florida, 33620, United States.
  • 2 Johns Hopkins University, School of Medicine, Baltimore, MD, 21205, USA.
  • 3 Department of Molecular Medicine, University of South Florida, Tampa, Florida, 33612, United States.
  • 4 Moffitt Cancer Center, Tampa, FL, 33612, United States.
  • 5 University of Florida, College of Medicine, Gainesville, FL, 32610-0216, United States.
  • 6 Florida Center of Excellence for Drug Discovery and Innovation, 3720 Spectrum Boulevard, Suite 305, Tampa, FL, 33612, United States.
  • 7 Department of Molecular Medicine, University of South Florida, Tampa, Florida, 33612, United States. ychen1@health.usf.edu.
  • 8 Department of Chemistry, University of South Florida, Tampa, Florida, 33620, United States. merkler@usf.edu.
PMID: 29044148 DOI: 10.1038/s41598-017-13669-6
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.

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