title: Purification, properties, and sequence of glycerol trinitrate reductase from Agrobacterium radiobacter.
abstract: Glycerol trinitrate (GTN) reductase, which enables Agrobacterium radiobacter to utilize GTN and related explosives as sources of nitrogen for growth, was purified and characterized, and its gene was cloned and sequenced. The enzyme was a 39-kDa monomeric protein which catalyzed the NADH-dependent reductive scission of GTN (Km = 23 microM) to glycerol dinitrates (mainly the 1,3-isomer) with a pH optimum of 6.5, a temperature optimum of 35 degrees C, and no dependence on metal ions for activity. It was also active on pentaerythritol tetranitrate (PETN), on isosorbide dinitrate, and, very weakly, on ethyleneglycol dinitrate, but it was inactive on isopropyl nitrate, hexahydro-1,3,5-trinitro-1,3,5-triazine, 2,4,6-trinitrotoluene, ammonium ions, nitrate, or nitrite. The amino acid sequence deduced from the DNA sequence was homologous (42 to 51% identity and 61 to 69% similarity) to those of PETN reductase from Enterobacter cloacae, N-ethylmaleimide reductase from Escherichia coli, morphinone reductase from Pseudomonas putida, and old yellow enzyme from Saccharomyces cerevisiae, placing the GTN reductase in the alpha/beta barrel flavoprotein group of proteins. GTN reductase and PETN reductase were very similar in many respects except in their distinct preferences for NADH and NADPH cofactors, respectively.
authors.affiliation: School of Molecular and Medical Biosciences, University of Wales Cardiff, United Kingdom.
authors.forename: J R
authors.initials: JR
authors.lastname: Snape
authors.forename: N A
authors.initials: NA
authors.lastname: Walkley
authors.forename: A P
authors.initials: AP
authors.lastname: Morby
authors.forename: S
authors.initials: S
authors.lastname: Nicklin
authors.forename: G F
authors.initials: GF
authors.lastname: White
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D012231:Rhizobium / Q000201:enzymology*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D010088:Oxidoreductases; C077366:glyceryl trinitrate reductase