Characterization and inactivation of an agmatine deiminase from Helicobacter pylori.
Helicobacter pylori encodes a potential virulence factor, agmatine deiminase (HpAgD), which catalyzes the conversion of agmatine to N-carbamoyl putrescine (NCP) and ammonia - agmatine is decarboxylated arginine. Agmatine is an endogenous human cell signaling molecule that triggers the innate immune response in humans. Unlike H. pylori, humans do not encode an AgD; it is hypothesized that inhibition of this enzyme would increase the levels of agmatine, and thereby enhance the innate immune response. Taken together, these facts suggest that HpAgD is a potential drug target. Herein we describe the optimized expression, isolation, and purification of HpAgD (10-30 mg/L media). The initial kinetic characterization of this enzyme has also been performed. Additionally, the crystal structure of wild-type HpAgD has been determined at 2.1A resolution. This structure provides a molecular basis for the preferential deimination of agmatine, and identifies Asp198 as a key residue responsible for agmatine recognition, which has been confirmed experimentally. Information gathered from these studies led to the development and characterization of a novel class of haloacetamidine-based HpAgD inactivators. These compounds are the most potent AgD inhibitors ever described.
Jones
Justin E
JE
Department of Chemistry & Biochemistry, University of South Carolina, 631 Sumter Street, Columbia, SC 29208, United States.
Causey
Corey P
CP
Lovelace
Leslie
L
Knuckley
Bryan
B
Flick
Heather
H
Lebioda
Lukasz
L
Thompson
Paul R
PR
D000376:Agmatine / Q000276:immunology / Q000378:metabolism; D000578:Amidines / Q000737:chemistry / Q000494:pharmacology; D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D016480:Helicobacter pylori / Q000201:enzymology*; D006801:Humans; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D000578:Amidines; D011994:Recombinant Proteins; D000376:Agmatine; D006867:Hydrolases; C019626:agmatine deiminase; C023731:acetamidine