Enzymes and Metabolites in Picric Acid (2,4,6-Trinitrophenol) and 2,4-Dinitrophenol Biodegradation

5 Abstract The bacterial strains Rhodococcus (opacus) erythropolis HL PM-1 and Nocardioides simplex FJ2-1A can use picric acid (TNP) and 2,4-dinitrophenol (DNP) as sole nitrogen source. Enzymes were identified which are isofunctional in both strains, using the same degradation mechanism. During the initial steps hydride ions were transferred to the aromatic ring of the electron deficient π-system, forming first the hydride σ-complex of TNP, H-TNP, and subsequently the dihydride σ-complex, 2H-TNP. The reactions were catalyzed by hydride transferase II (HTII) and hydride transferase I (HTI), respectively, together with the NADPH-dependent F420 reductase (NDFR) and the coenzymes NADPH and F420. NDFR, HTII, and HTI were cloned and purified as His-tag fusion proteins by NiNTA affinity chromatography. Reactions were followed by repeated recording of UVvisible spectra, and intermediates were identified by HPLC, HPLC-MS, and NMR measurements. H-TNP and 2H-TNP were chemically synthesized and used as standards and substrates. In the next step, a nitrite-eliminating enzyme releasing nitrite from 2H-TNP and generating the hydride σ-complex of DNP (H-DNP) was identified. A tautomerase was shown to give rise to two tautomeric forms, the aci-nitro form and the nitro form of 2H-TNP, identified by HPLC and NMR measurements. Since the nitrite-eliminating enzyme used only the aci-nitro form as a substrate, it appears that the 2H-TNP tautomerase inhibits accumulation of the nitro form. Whereas the 2H-TNP tautomerase was cloned and purified as a His-tag fusion protein, the nitriteeliminating enzyme was isolated by FPLC and the N-terminal sequence was determined. Databank search exhibited no similarities to any known proteins. HTI and NDFR, together with the coenzymes NADPH and F420, hydrogenated chemically synthesized H-DNP to 2H-DNP. It was shown by HPLC-MS that 2,4dinitrocyclohexanone (2,4-DNCH) was generated after protonation. A hydrolase was purified by FPLC, catalyzing hydrolytic ring fission of 2,4-DNCH, forming 4,6dinitrohexanoate (4,6-DNH). 4,6-DNH was identified by HPLC-MS. MALDI-TOF measurements showed that the 2,4-DNCH hydrolase consists of four identical subunits. Since determination of the N-terminal amino acid sequence exhibited thatThe bacterial strains Rhodococcus (opacus) erythropolis HL PM-1 and Nocardioides simplex FJ2-1A can use picric acid (TNP) and 2,4-dinitrophenol (DNP) as sole nitrogen source. Enzymes were identified which are isofunctional in both strains, using the same degradation mechanism. During the initial steps hydride ions were transferred to the aromatic ring of the electron deficient π-system, forming first the hydride σ-complex of TNP, H-TNP, and subsequently the dihydride σ-complex, 2H-TNP. The reactions were catalyzed by hydride transferase II (HTII) and hydride transferase I (HTI), respectively, together with the NADPH-dependent F420 reductase (NDFR) and the coenzymes NADPH and F420. NDFR, HTII, and HTI were cloned and purified as His-tag fusion proteins by NiNTA affinity chromatography. Reactions were followed by repeated recording of UVvisible spectra, and intermediates were identified by HPLC, HPLC-MS, and NMR measurements. H-TNP and 2H-TNP were chemically synthesized and used as standards and substrates. In the next step, a nitrite-eliminating enzyme releasing nitrite from 2H-TNP and generating the hydride σ-complex of DNP (H-DNP) was identified. A tautomerase was shown to give rise to two tautomeric forms, the aci-nitro form and the nitro form of 2H-TNP, identified by HPLC and NMR measurements. Since the nitrite-eliminating enzyme used only the aci-nitro form as a substrate, it appears that the 2H-TNP tautomerase inhibits accumulation of the nitro form. Whereas the 2H-TNP tautomerase was cloned and purified as a His-tag fusion protein, the nitriteeliminating enzyme was isolated by FPLC and the N-terminal sequence was determined. Databank search exhibited no similarities to any known proteins. HTI and NDFR, together with the coenzymes NADPH and F420, hydrogenated chemically synthesized H-DNP to 2H-DNP. It was shown by HPLC-MS that 2,4dinitrocyclohexanone (2,4-DNCH) was generated after protonation. A hydrolase was purified by FPLC, catalyzing hydrolytic ring fission of 2,4-DNCH, forming 4,6dinitrohexanoate (4,6-DNH). 4,6-DNH was identified by HPLC-MS. MALDI-TOF measurements showed that the 2,4-DNCH hydrolase consists of four identical subunits. Since determination of the N-terminal amino acid sequence exhibited that