The kinase activity of the Helicobacter pylori Asp-tRNA(Asn)/Glu-tRNA(Gln) amidotransferase is sensitive to distal mutations in its putative ammonia tunnel.
The Helicobacter pylori (Hp) Asp-tRNA(Asn)/Glu-tRNA(Gln) amidotransferase (AdT) plays important roles in indirect aminoacylation and translational fidelity. AdT has two active sites, in two separate subunits. Kinetic studies have suggested that interdomain communication occurs between these subunits; however, this mechanism is not well understood. To explore domain-domain communication in AdT, we adapted an assay and optimized it to kinetically characterize the kinase activity of Hp AdT. This assay was applied to the analysis of a series of point mutations at conserved positions throughout the putative AdT ammonia tunnel that connects the two active sites. Several mutations that caused significant decreases in AdT's kinase activity (reduced by 55-75%) were identified. Mutations at Thr149 (37 Å distal to the GatB kinase active site) and Lys89 (located at the interface of GatA and GatB) were detrimental to AdT's kinase activity, suggesting that these mutations have disrupted interdomain communication between the two active sites. Models of wild-type AdT, a valine mutation at Thr149, and an arginine mutation at Lys89 were subjected to molecular dynamics simulations. A comparison of wild-type, T149V, and K89R AdT simulation results unmasks 59 common residues that are likely involved in connecting the two active sites.
Department of Chemistry, Wayne State University, Detroit, Michigan 48202, United States.
Liangjun
L
Zhao
Sajeewa W
SW
Dewage
Michael J
MJ
Bell
Keng-Ming
KM
Chang
Shirin
S
Fatma
Nilesh
N
Joshi
Gayathri
G
Silva
G Andrés
GA
Cisneros
Tamara L
TL
Hendrickson
D000641:Ammonia / Q000737:chemistry*; D001216:Asparagine / Q000235:genetics; D001226:Aspartate-tRNA Ligase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D005973:Glutamine / Q000096:biosynthesis / Q000172:deficiency*; D016480:Helicobacter pylori / Q000201:enzymology* / Q000235:genetics; D008239:Lysine / Q000235:genetics; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D019884:Nitrogenous Group Transferases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D010766:Phosphorylation / Q000235:genetics; D012346:RNA, Transfer, Amino Acyl / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D013211:Staphylococcus aureus / Q000201:enzymology / Q000235:genetics; D014443:Tyrosine / Q000235:genetics
D012346:RNA, Transfer, Amino Acyl; D005973:Glutamine; D014443:Tyrosine; D001216:Asparagine; D000641:Ammonia; C412016:Asp-tRNA(Asn) amidotransferase; D019884:Nitrogenous Group Transferases; D001226:Aspartate-tRNA Ligase; C029194:asparaginyl-tRNA synthetase; D008239:Lysine