Biochemical characteristics of a nitroreductase with diverse substrate specificity from Streptomyces mirabilis DUT001

Abstract

A nitroreductase-encoded gene from an efficient nitro-reducing bacterium Streptomyces mirabilis DUT001, named snr, was cloned and heterogeneously expressed in Escherichia coli. The purified Streptomyces nitroreductase SNR was a homodimer with an apparent subunit molecular weight of 24 kDa and preferred NADH to NADPH as a cofactor. By enzyme incubation and isothermal calorimetry experiments, flavin mononucleotide (FMN) was found to be the preferred flavin cofactor; the binding process was exothermic and primarily enthalpy driven. The enzyme can reduce multiple nitro compounds and flavins, including antibacterial drug nitrofurazone, priority pollutants 2,4-dinitrotoluene and 2,4,6-trinitrotoluene, as well as key chemical intermediates 3-nitrophthalimide, 4-nitrophthalimide, and 4-nitro-1,8-naphthalic anhydride. Among the substrates tested, the highest activity of k /K (0.234 μM Sec ) was observed for the reduction of FMN. Multiple sequence alignment revealed that the high FMN reduction activity of SNR may be due to the absence of a helix, constituting the entrance to the substrate pocket in other nitroreductases.

Synthesis of Pharmaceutically Relevant Arylamines Enabled by a Nitroreductase from Bacillus tequilensis

“…Based on literature data and a genome mining approach, four nitroreductase-like flavoenzymes were initially selected for this study (see Supporting Information for details). Three of these flavoenzymes are well-characterized NRs that have been reported to perform amine synthesis starting from nitro aromatic compounds, namely NfnB from Mycobacterium smegmatis, [16,22] SNR from Streptomyces mirabilis [26,31] and NfnB from Sphingopyxis sp. [32] Additionally, a putative NR from Bacillus tequilensis, named BtNR, was also selected (Figure S1).…”

“…Therefore, the ability to perform the full nitro reduction to the respective amine depends not only on the prosthetic flavin's redox potential but also on many other factors. The majority of well‐characterized NRs mainly produce hydroxylamines, with a few exceptions forming amines in low yields, such as NfnB from Mycobacterium smegmatis , [16] SNR from Streptomyces mirabilis , [26] NRSal from Salmonella typhimurium , [17] Gox0834 from Gluconobacter oxydans 621H , and two NRs from Klebsiella sp. C1 [27] .…”

2D Strategy for the Construction of an Enzyme-Activated NIR Fluorophore Suitable for the Visual Sensing and Profiling of Homologous Nitroreductases from Various Bacterial Species

“…Nitroreductases (NTRs) are biological enzymes of the flavin enzyme family that reduce nitroaromatic compounds to the corresponding nitrite, hydroxylamine, or amino derivatives using nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH) as an electron donor. − The hypoxic environment of the tumor tissue results in the overexpression of NTRs in tumor cells, highlighting the importance of NTR monitoring for clinical diagnosis and tumor therapy. , Compared with the role of NTRs in mammalian cells, bacterial NTRs are thought to play a vital role in the antibacterial activity of nitroimidazole antibiotics, such as chloramphenicol. − In bacterial cells, intracellular reduction of the nitro groups of 5-nitroimidazole drugs (metronidazole, tinidazole, and ornidazole) can be mediated by endogenous NTRs along with the production of active radical intermediates, which inhibits bacterial colonization through the inhibition of DNA synthesis. Clinically, emerging problems of resistance to 5-nitroimidazole drugs make the treatment of bacterial infections a growing challenge .…”