Overexpression, Isotopic Labeling, and Spectral Characterization ofEnterobacter cloacaeNitroreductase

Koder, Ronald L.; Miller, Anne‐Frances

doi:10.1006/prep.1997.0866

Cited by 18 publications

(24 citation statements)

References 40 publications

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“…Fully flavinated NR was identified based on a ratio of A 280 /A 454 < 4.5. The concentration of holo NR was estimated using the extinction coefficient, ε 454 = 14.3 × 10 3 M −1 cm −1 (Koder and Miller, 1998b). .…”

Section: Star Methodsmentioning

confidence: 99%

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

et al. 2017

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Summary Nitroreductase from Enterobacter cloacae (NR) reduces diverse nitroaromatics including herbicides, explosives and prodrugs, and holds promise for bioremediation, prodrug activation and enzyme-assisted synthesis. We solved crystal structures of NR complexes with bound substrate or analog for each of its two half-reactions. We complemented these with kinetic isotope effect (KIE) measurements elucidating H-transfer steps essential to each half-reaction. KIEs indicate hydride transfer from NADH to the flavin consistent with our structure of NR with the NADH analog nicotinic acid adenine dinucleotide (NAAD). The KIE on reduction of p-nitrobenzoic acid (p-NBA) also indicates hydride transfer, and requires revision of prior computational mechanisms. Our mechanistic information provided a structural restraint for the orientation of bound substrate, placing the nitro group closer to the flavin N5 in the pocket that binds the amide of NADH. KIEs show that solvent provides a proton, enabling accommodation of different nitro group placements, consistent with NR’s broad repertoire.

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Section: Star Methodsmentioning

confidence: 99%

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

et al. 2017

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“…The stability and purity of p-HABA in anaerobic versus airequilibrated buffer was also characterized by 1 H NMR spectroscopy at 400 MHz. p-HABA (13.3 mM) was dissolved in 50 mM potassium phosphate, pH 7.50, containing 10% v/v 2 H 2 O and 0.1 mM 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) as a chemical shift reference (␦ ϭ 0 ppm).…”

Section: Methodsmentioning

confidence: 99%

Understanding the Broad Substrate Repertoire of Nitroreductase Based on Its Kinetic Mechanism

Pitsawong

Hoben

Miller

2014

Journal of Biological Chemistry

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Background: Nitroreductase reduces a broad range of nitroaromatics. Results: Steady-state and pre-steady-state kinetics were combined with tests for aminoaromatic product formation. Conclusion: Both half-reactions occur via a simple mechanism lacking detectable gating steps consistent with the broad substrate repertoire. Significance: Nitroreductase does not generate p-aminobenzoic acid and, therefore, appears not to reduce nitro groups to amines.

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“…The nitroreductase NR from Enterobacter cloacae is a homodimer, although it is also active as a 27 kDa monomer, and shows apparent K m values for TNT and NADPH of 56 and 443 μM, respectively (Bryant & DeLuca, 1991; Koder & Miller, 1998). Sequence analysis of the gene coding for the NR nitroreductase of Enterobacter cloacae (Bryant et al , 1991) and the RNR nitroreductase gene from an Enterobacter strain isolated from a patient (Koder et al , 2001) indicates that these nitroreductases are closely related to the Escherichia coli NfsB nitroreductase (Fig.…”

Section: Bacterial Nitroreductasesmentioning

confidence: 99%

Reduction of polynitroaromatic compounds: the bacterial nitroreductases

et al. 2008

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Most nitroaromatic compounds are toxic and mutagenic for living organisms, but some microorganisms have developed oxidative or reductive pathways to degrade or transform these compounds. Reductive pathways are based either on the reduction of the aromatic ring by hydride additions or on the reduction of the nitro groups to hydroxylamino and/or amino derivatives. Bacterial nitroreductases are flavoenzymes that catalyze the NAD(P)H-dependent reduction of the nitro groups on nitroaromatic and nitroheterocyclic compounds. Nitroreductases have raised a great interest due to their potential applications in bioremediation, biocatalysis, and biomedicine, especially in prodrug activation for chemotherapeutic cancer treatments. Different bacterial nitroreductases have been purified and their biochemical and kinetic parameters have been determined. The crystal structure of some nitroreductases have also been solved. However, the physiological role(s) of these enzymes remains unclear. Nitroreductase genes are widely spread within bacterial genomes, but are also found in archaea and some eukaryotic species. Although studies on regulation of nitroreductase gene expression are scarce, it seems that nitroreductase genes may be controlled by the MarRA and SoxRS regulatory systems that are involved in responses to several antibiotics and environmental chemical hazards and to specific oxidative stress conditions. This review covers the microbial distribution, types, biochemical properties, structure and regulation of the bacterial nitroreductases. The possible physiological functions and the biotechnological applications of these enzymes are also discussed.

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Overexpression, Isotopic Labeling, and Spectral Characterization ofEnterobacter cloacaeNitroreductase

Cited by 18 publications

References 40 publications

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

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

Understanding the Broad Substrate Repertoire of Nitroreductase Based on Its Kinetic Mechanism

Reduction of polynitroaromatic compounds: the bacterial nitroreductases

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