npd gene functions of Rhodococcus (opacus) erythropolis HL PM-1 in the initial steps of 2,4,6-trinitrophenol degradation.
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.
Heiss
Gesche
G
Institute of Microbiology, University of Stuttgart, Allmandring 31, 70550 Stuttgart, Germany1.
Hofmann
Klaus W
KW
Institute of Microbiology, University of Stuttgart, Allmandring 31, 70550 Stuttgart, Germany1.
Trachtmann
Natalie
N
Institute of Microbiology, University of Stuttgart, Allmandring 31, 70550 Stuttgart, Germany1.
Walters
Dana M
DM
DuPont de Nemours Company, Wilmington, Delaware, USA2.
Rouvière
Pierre
P
DuPont de Nemours Company, Wilmington, Delaware, USA2.
Knackmuss
Hans-Joachim
HJ
Institute of Microbiology, University of Stuttgart, Allmandring 31, 70550 Stuttgart, Germany1.
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
D001426:Bacterial Proteins; D010851:Picrates; C005858:picric acid; D010088:Oxidoreductases; D009247:NADH, NADPH Oxidoreductases; C109941:F420-dependent NADP reductase; D014166:Transferases