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npd gene functions of Rhodococcus (opacus) erythropolis HL PM-1 in the initial steps of 2,4,6-trinitrophenol degradation^b

Institute of Microbiology, University of Stuttgart, Allmandring 31, 70550 Stuttgart, Germany¹
DuPont de Nemours Company, Wilmington, Delaware, USA²

Author for correspondence: Gesche Heiss. Tel: +49 711 685 5491. Fax: +49 711 685 5725. e-mail: gesche.heiss{at}po.uni-stuttgart.de

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 -complex (hydride Meisenheimer complex) of picric acid (H^--PA). Similarly, NpdI also transformed 2,4-DNP to the hydride -complex of 2,4-DNP. A second hydride transferase, NpdC catalysed a subsequent hydride transfer to H^--PA, to produce a dihydride -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.

Keywords: picric acid, two hydride transferases, NADPH-dependent F₄₂₀ reductase

Abbreviations: H^--PA, hydride -complex of picric acid; 2H^--PA, dihydride -complex of picric acid; 2,4-DNP, 2,4-dinitrophenol; H^--2,4-DNP, hydride -complex of 2,4-dinitrophenol; HTES, hydride transferring enzyme system

^b The GenBank accession number for the sequence reported in this paper is AF435009.

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