title: npd gene functions of Rhodococcus (opacus) erythropolis HL PM-1 in the initial steps of 2,4,6-trinitrophenol degradation.
abstract: Rhodococcus (opacus) erythropolis HL PM-1 grows on 2,4,6-trinitrophenol (picric acid) or 2,4-dinitrophenol (2,4-DNP) as sole nitrogen source. A gene cluster involved in picric acid degradation was recently identified. The functional assignment of three of its genes, npdC, npdG and npdI, and the tentative functional assignment of a fourth one, npdH, is reported. The genes were expressed in Escherichia coli as His-tag fusion proteins that were purified by Ni-affinity chromatography. The enzyme activity of each protein was determined by spectrophotometry and HPLC analyses. NpdI, a hydride transferase, catalyses a hydride transfer from reduced F420 to the aromatic ring of picric acid, generating the hydride sigma-complex (hydride Meisenheimer complex) of picric acid (H(-)-PA). Similarly, NpdI also transformed 2,4-DNP to the hydride sigma-complex of 2,4-DNP. A second hydride transferase, NpdC catalysed a subsequent hydride transfer to H(-)-PA, to produce a dihydride sigma-complex of picric acid (2H(-)-PA). All three reactions required the activity of NpdG, an NADPH-dependent F420 reductase, for shuttling the hydride ions from NADPH to F420. NpdH converted 2H(-)-PA to a hitherto unknown product, X. The results show that npdC, npdG and npdI play a key role in the initial steps of picric acid degradation, and that npdH may prove to be important in the later stages.
authors.affiliation: Institute of Microbiology, University of Stuttgart, Allmandring 31, 70550 Stuttgart, Germany1.
authors.forename: Gesche
authors.initials: G
authors.lastname: Heiss
authors.affiliation: Institute of Microbiology, University of Stuttgart, Allmandring 31, 70550 Stuttgart, Germany1.
authors.forename: Klaus W
authors.initials: KW
authors.lastname: Hofmann
authors.affiliation: Institute of Microbiology, University of Stuttgart, Allmandring 31, 70550 Stuttgart, Germany1.
authors.forename: Natalie
authors.initials: N
authors.lastname: Trachtmann
authors.affiliation: DuPont de Nemours Company, Wilmington, Delaware, USA2.
authors.forename: Dana M
authors.initials: DM
authors.lastname: Walters
authors.affiliation: DuPont de Nemours Company, Wilmington, Delaware, USA2.
authors.forename: Pierre
authors.initials: P
authors.lastname: Rouvière
authors.affiliation: Institute of Microbiology, University of Stuttgart, Allmandring 31, 70550 Stuttgart, Germany1.
authors.forename: Hans-Joachim
authors.initials: HJ
authors.lastname: Knackmuss
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D010851:Picrates / Q000378:metabolism*; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics; D014166:Transferases / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D010851:Picrates; C005858:picric acid; D010088:Oxidoreductases; D009247:NADH, NADPH Oxidoreductases; C109941:F420-dependent NADP reductase; D014166:Transferases