Characterization of a Nitroreductase with Selective Nitroreduction Properties in the Food and Intestinal Lactic Acid Bacterium Lactobacillus plantarum WCFS1

Abstract

Nitroreductases reduce nitroaromatic compounds and other oxidants in living organisms, having interesting implications in environmental and human health. A putative nitrobenzoate reductase encoding gene (lp_0050) was recently annotated in the completed DNA sequence of lactic acid bacterium Lactobacillus plantarum WCFS1 strain. In this research, this L. plantarum gene was cloned and expressed, and the corresponding protein (PnbA) was biochemically characterized. This L. plantarum PnbA reductase is a 216 amino acid residue FMN-flavoprotein, which exhibits 23% identity with Pseudomonas putida and Ralstonia eutropha nitroreductases and <11% identity with those from enterobacteria such as E. cloacae . This reductase also showed 32-43% identity (65-72% similarity) to predicted PnbA proteins from other lactic acid bacteria. It utilized a wide range of electron acceptors including dichlorophenolindophenol (DCPIP), nitroblue tetrazolium (NBT), ferricyanide, and quinones (menadione, benzoquinone), but not pyridinium cations (paraquat and N-methyl-beta-carbolines), and it was inhibited by dicoumarol and diphenyliodonium. HPLC-MS and spectroscopic data showed that it specifically catalyzed the reduction of the 4-nitroaromatic group to the corresponding hydroxylamine in the presence of NAD(P)H. Kinetics parameters (V(max) and K(m)) showed a higher efficiency for the reduction of 2,4-dinitrobenzoate than for the reduction of 4-nitrobenzoate. It was chemoselective for the reduction of 4-nitrobenzoates, being unable to reduce other nitroaromatics. Then, L. plantarum PnbA reductase might be more specific than other microbial nitroreductases that reduce a wider range of nitroaromatic compounds. The physiological and functional role of nitroreductases remain unknown; however, their presence in lactic acid bacteria widely occurring in foods and the human intestinal tract should be of further interest.

Taiwanofungus camphorata nitroreductase: cDNA cloning and biochemical characterisation

“…The results suggest that a histidine residue (in or near the active site) is involved in the catalytic activity because side chain's pKa of histitine is 6.0. It should be noted that most of the Nr activity assays reported in this paper were performed at pH 7.0 similar to that reported by Guillen et al (2009). Thus the TcNr would be more efficient in reducing nitrocompounds at pH 6.0.…”

“…Nr activity was determined by measuring NADPH dependent reduction of potassium ferricyanide, K 3 [Fe(CN) 6 ] (Guillen, Curiel, Landete, Munoz, & Herraiz, 2009). A typical 100 lL reaction mixture contained 10 mM Tris-Cl (pH 7.0), 1 mM NADPH and 0.1-2 mM ferricyanide.…”

Mechanism-Informed Refinement Reveals Altered Substrate-Binding Mode for Catalytically Competent Nitroreductase

“…NR’s action is essential to the efficacy of widely-used antibiotics such as metronidazole and to treatments for stomach ulcers and dysentery (Pal et al, 2009). NRs of gut flora convert nitroaromatic compounds to more toxic products, also raising the incidence of cancer (Guillén et al, 2009). However ongoing efforts to harness NR’s activity to combat cancer demonstrate NR’s promise as an agent for enzymatic activation of prodrugs in therapies and diagnostics (Williams et al, 2015).…”

Pendimethalin Nitroreductase Is Responsible for the Initial Pendimethalin Degradation Step in Bacillus subtilis Y3

“…Subfamily B1 includes the minor nitroreductase NfsB of E. coli (33), the retro-nitroreductase (RNR) of Enterobacter cloacae (34), the nitroreductase PnrB of P. putida JLR11 (35), the NAD(P)H-flavin oxidoreductase FRase I of V. fischeri (36), the quinone reductase DrgA of Synechocystis sp. (37), the nitrobenzoate nitroreductase PnbA of L. plantarum WCFS1 (38), and others. Subfamily B2 contains the YdjA of E. coli and Salmonella enterica and the nitrobenzene nitroreductase CnbA encoded by the cnbA gene located on plasmid pCNB1 of Comamonas testosteroni CNB-1 (39).…”