PnrA, a new nitroreductase‐family enzyme in the TNT‐degrading strain Pseudomonas putida JLR11

Abstract

Nitroreductases are a group of proteins that catalyse pyridine nucleotide-dependent reduction of nitroaromatics compounds, showing significant human health and environmental implications. In this study we have identified the nitroreductase-family enzymes PnrA and PnrB from the TNT-degrading strain Pseudomonas putida. The enzyme encoded by the pnrA gene was expressed in Escherichia coli, purified to homogeneity and shown to be a flavoprotein that used 2 mol of NADPH to reduce 1 mol of 2,4,6-trinitrotoluene (TNT) to 4-hydroxylamine-2,6-dinitrotoluene, using a ping-pong bi-bi mechanism. The PnrA enzyme also recognized as substrates as a number of other nitroaromatic compounds, i.e. 2,4-dinitrotoluene, 3-nitrotoluene, 3- and 4-nitrobenzoate, 3,5-dinitrobenzamide and 3,5-dinitroaniline expanding the substrates profile from previously described nitroreductases. However, TNT resulted to be the most efficient substrate examined according to the Vmax/Km parameter. Expression analysis of pnrA- and pnrB-mRNA isolated from cells growing on different nitrogen sources suggested that expression of both genes was constitutive and that its level of expression was relatively constant regardless of the growth substrate. This is in agreement with enzyme-specific activity determined with cells growing with different N-sources.

Characterization of a Pseudomonad 2-Nitrobenzoate Nitroreductase and Its Catabolic Pathway-Associated 2-Hydroxylaminobenzoate Mutase and a Chemoreceptor Involved in 2-Nitrobenzoate Chemotaxis

“…For the first time, a homology model of 2-NBA nitroreductase is available, which adds to our limited knowledge on the structure and function of nitroreductases in nitroaromatic metabolism for which a limited number of ge- (11,35,40,51,69). NbaA is a newly discovered representative of a family of homodimeric NADH:FMN oxidoreductase-like fold proteins that is different from the nitro/flavin reductase family.…”

Nitroaromatic Compounds, from Synthesis to Biodegradation

“…The nitroreductases NfsA and NfsB, together with the N-ethylmaleimide reductase NemA, contributed to the ability of E. coli AB1157 to obtain usable nitrogen from TNT (51). Similarly, the nitroreductase PrnA was shown to be involved in the utilization of TNT as a nitrogen source by P. putida JLR11, and the assimilatory nitrite reductase NasB contributed to the ability of the strain to grow efficiently (14,15). A mechanism for the release of nitrite during the condensation of hydroxylaminodinitrotoluene (the product of nitroreductase activity on TNT) and a Meisenheimer dihydride complex (Fig.…”

“…Two Gram-positive isolates, strains TNT-8 and TNT-32, were able to use TNT as a nitrogen source, but the mechanism of nitrogen assimilation remains unclear (200). More recently, Ramos et al reported that Pseudomonas putida JLR11 (13)(14)(15) and Escherichia coli AB1157 (51) were able to use TNT as a nitrogen source for growth, reducing the nitro group and recovering the ammonium by the use of nitroreductases. The nitroreductases NfsA and NfsB, together with the N-ethylmaleimide reductase NemA, contributed to the ability of E. coli AB1157 to obtain usable nitrogen from TNT (51).…”

Subfunctionality of Hydride Transferases of the Old Yellow Enzyme Family of Flavoproteins of Pseudomonas putida

“…These compounds are susceptible to the reduction of the nitro side groups to yield hydroxylamino groups. This type of activity is also called nitroreductase activity (6,7,8,9,20,28,34) and will be referred to here as type I hydride transferase activity. Other OYE members have been described to additionally catalyze the nucleophilic attack on the aromatic ring of 2,4,6-trinitrotoluene (TNT) by hydride ions to produce Meisenheimer monohydride and dihydride complexes (13,23,24,29,32,37).…”