title: Characterization of tunnel mutants reveals a catalytic step in ammonia delivery by an aminoacyl-tRNA amidotransferase.
abstract: The Helicobacter pylori Asp-tRNA(A) (sn) /Glu-tRNA(G) (ln) amidotransferase (GatCAB) utilizes an uncommonly hydrophilic, ~ 40 Å ammonia tunnel for ammonia/ammonium transport between isolated active sites. Hydrophilicity of this tunnel requires a distinct ammonia transport mechanism, which hypothetically occurs through a series of deprotonation and protonation steps. To explore the initiation of this relay mechanism, the highly conserved tunnel residue D185 (in the GatA subunit) was enzymatically and computationally investigated by comparing D185A, D185N, and D185E mutant enzymes to wild-type GatCAB. Our results indicate that D185 acts as an acid/base residue, participating directly in catalysis. To our knowledge, this is the first example of acid/base chemistry in a glutamine-dependent amidotransferase ammonia tunnel.
authors: Department of Chemistry, Wayne State University, Detroit, MI, USA.
authors: Liangjun
authors: L
authors: Zhao
authors: Department of Chemistry, Wayne State University, Detroit, MI, USA.
authors: Udumbara M
authors: UM
authors: Rathnayake
authors: Department of Chemistry, Wayne State University, Detroit, MI, USA.
authors: Sajeewa W
authors: SW
authors: Dewage
authors: Department of Chemistry, Wayne State University, Detroit, MI, USA.
authors: Whitney N
authors: WN
authors: Wood
authors: Department of Chemistry, Wayne State University, Detroit, MI, USA.
authors: Anthony J
authors: AJ
authors: Veltri
authors: Department of Chemistry, Wayne State University, Detroit, MI, USA. Andres@chem.wayne.edu.
authors: G Andrés
authors: GA
authors: Cisneros
authors: Department of Chemistry, Wayne State University, Detroit, MI, USA. Tamara.Hendrickson@chem.wayne.edu.
authors: Tamara L
authors: TL
authors: Hendrickson
mesh_terms: D000641:Ammonia / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D016480:Helicobacter pylori / Q000201:enzymology*; D056004:Molecular Dynamics Simulation; D020125:Mutation, Missense; D019884:Nitrogenous Group Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D000641:Ammonia; C525150:GatCAB amidotransferase, Helicobacter pylori; D019884:Nitrogenous Group Transferases
keywords: AdT; GatCAB; ammonia tunnel; correlation studies; glutamine-dependent amidotransferase; molecular dynamics; tRNA transamidation