title: An unusual tryptophanyl tRNA synthetase interacts with nitric oxide synthase in Deinococcus radiodurans.
abstract: In mammals, nitric oxide synthases (NOSs) produce nitric oxide for signaling and defense functions; in Streptomyces, NOS proteins nitrate a tryptophanyl moiety in synthesis of a phytotoxin. We have discovered that the NOS protein from the radiation-resistant bacterium Deinococcus radiodurans (deiNOS) associates with an unusual tryptophanyl tRNA synthetase (TrpRS). D. radiodurans contains genes for two TrpRSs: the first has approximately 40% sequence identity to typical TrpRSs, whereas the second, identified as the NOS-interacting protein (TrpRS II), has only approximately 29% identity. TrpRS II is induced after radiation damage and contains an N-terminal extension similar to those of proteins involved in stress responses. Recombinantly expressed TrpRS II binds tryptophan (Trp), ATP, and D. radiodurans tRNA(Trp) and catalyzes the formation of 5' adenyl-Trp and tRNA(Trp), with approximately five times less activity than TrpRS I. Upon coexpression in Escherichia coli, TrpRS II binds to, copurifies with, and dramatically enhances the solubility of deiNOS. Dimeric TrpRS II binds dimeric deiNOS with a stoichiometry of 1:1 and a dissociation constant of 6-30 muM. Upon forming a complex, deiNOS quenches the fluorescence of an ATP analog bound to TrpRS II, and increases its affinity for substrate l-arginine. Remarkably, TrpRS II also activates the NOS activity of deiNOS. These findings reveal a link between bacterial NOS and Trp metabolism in a second organism and may indicate yet another novel biological function for bacterial NOS.
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Madhavan R
authors.initials: MR
authors.lastname: Buddha
authors.forename: Kim M
authors.initials: KM
authors.lastname: Keery
authors.forename: Brian R
authors.initials: BR
authors.lastname: Crane
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000595:Amino Acid Sequence; D034301:Deinococcus / Q000201:enzymology* / Q000235:genetics; D019281:Dimerization; D005798:Genes, Bacterial; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012364:RNA, Transfer, Trp / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D014369:Tryptophan-tRNA Ligase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D007527:Isoenzymes; D012364:RNA, Transfer, Trp; D011994:Recombinant Proteins; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D019001:Nitric Oxide Synthase; D014369:Tryptophan-tRNA Ligase