The interaction of ammonia and xenon with the imidazole glycerol phosphate synthase from Thermotoga maritima as detected by NMR spectroscopy.
The imidazole glycerol phosphate (ImGP) synthase from the hyperthermophilic bacterium Thermotoga maritima is a 1:1 complex of the glutaminase subunit HisH and the cyclase subunit HisF. It has been proposed that ammonia generated by HisH is transported through a channel to the active site of HisF, which generates intermediates of histidine (ImGP) and de novo biosynthesis of 5-aminoimidazole-4-carboxamideribotide. Solution NMR spectroscopy of ammonium chloride-titrated samples was used to study the interaction of NH(3) with amino acids inside this channel. Although numerous residues showed (15)N chemical shift changes, most of these changes were caused by nonspecific ionic strength effects. However, several interactions appeared to be specific. Remarkably, the amino acid residue Thr 78-which is located in the central channel-shows a large chemical shift change upon titration with ammonium chloride. This result and the reduced catalytic activity of the Thr78Met mutant indicate a special role of this residue in ammonia channeling. To detect and further characterize internal cavities in HisF, which might for example contribute to ammonia channeling, the interaction of HisF with the noble gas xenon was analyzed by solution NMR spectroscopy using (1)H-(15)N HSQC experiments. The results indicate that HisF contains three distinct internal cavities, which could be identified by xenon-induced chemical shift changes of the neighboring amino acid residues. Two of these cavities are located at the active site at opposite ends of the substrate N'-[(5'-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) binding groove. The third cavity is located in the interior of the central β-barrel of HisF and overlaps with the putative ammonia transport channel.
Liebold
Christoph
C
Institute of Biophysics and Physical Biochemistry, University of Regensburg, Regensburg, Germany.
List
Felix
F
Kalbitzer
Hans Robert
HR
Sterner
Reinhard
R
Brunner
Eike
E
D000619:Aminohydrolases / Q000737:chemistry / Q000378:metabolism*; D000641:Ammonia / Q000378:metabolism*; D001665:Binding Sites; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D020124:Thermotoga maritima / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D014978:Xenon / Q000378:metabolism*
D014978:Xenon; D000641:Ammonia; C087227:imidazole glycerol phosphate synthase; D000619:Aminohydrolases