title: Determination of the formylglycinamide ribonucleotide amidotransferase ammonia pathway by combining 3D-RISM theory with experiment.
abstract: Molecular tunnels in enzyme systems possess variable architecture and are therefore difficult to predict. In this work, we design and apply an algorithm to resolve the pathway followed by ammonia using the bifunctional enzyme formylglycinamide ribonucleotide amidotransferase (FGAR-AT) as a model system. Though its crystal structure has been determined, an ammonia pathway connecting the glutaminase domain to the 30 Å distal FGAR/ATP binding site remains elusive. Crystallography suggested two purported paths: an N-terminal-adjacent path (path 1) and an auxiliary ADP-adjacent path (path 2). The algorithm presented here, RismPath, which enables fast and accurate determination of solvent distribution inside a protein channel, predicted path 2 as the preferred mode of ammonia transfer. Supporting experimental studies validate the identity of the path, and results lead to the conclusion that the residues in the middle of the channel do not partake in catalytic coupling and serve only as channel walls facilitating ammonia transfer.
authors.affiliation: †Department of Chemistry, Indian Institute of Technology, IIT-Bombay, Mumbai 400076, India.
authors.forename: Ajay S
authors.initials: AS
authors.lastname: Tanwar
authors.affiliation: ‡College of Life Sciences, Ritsumeikan University and Molecular Design Frontier Co. Ltd., Kyoto, Japan.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Sindhikara
authors.affiliation: ‡College of Life Sciences, Ritsumeikan University and Molecular Design Frontier Co. Ltd., Kyoto, Japan.
authors.forename: Fumio
authors.initials: F
authors.lastname: Hirata
authors.affiliation: †Department of Chemistry, Indian Institute of Technology, IIT-Bombay, Mumbai 400076, India.
authors.forename: Ruchi
authors.initials: R
authors.lastname: Anand
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000465:Algorithms; D000641:Ammonia / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D019733:Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D005998:Glycine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D008958:Models, Molecular; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012265:Ribonucleotides / Q000737:chemistry / Q000378:metabolism; D012486:Salmonella typhimurium / Q000737:chemistry* / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D012265:Ribonucleotides; C002824:phosphoribosyl-N-formylglycineamide; D000641:Ammonia; D000255:Adenosine Triphosphate; D019733:Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor; C022385:phosphoribosylformylglycinamidine synthetase; D005998:Glycine