Journal of Molecular Biology
Regular articleThe structure of Escherichia coli nitroreductase complexed with nicotinic acid: three crystal forms at 1.7 Å, 1.8 Å and 2.4 Å resolution1
Introduction
The nfsB/nfnB gene of Escherichia coli, encoding the minor, oxygen-insensitive nitroreductase (NTR), was originally identified by mutations conferring resistance to the nitrofuran antibiotics, nitrofurazone and nitrofurantoin.1, 2 NTR is a FMN-containing protein (217 amino acid residues per polypeptide chain) with oxidoreductase activity.3 Unusually, it can accept electrons from either NADH or NADPH. The reduced enzyme in turn reduces a broad range of substrates, that includes quinones such as menadione, and nitroaromatics. The latter group includes the nitrofuran anti-biotics, and certain prodrugs proposed for cancer therapy, e.g. 5-{aziridin-1-yl}-2,4-dinitrobenzamide (CB1954).4, 5, 6
NTR has no significant sequence homology to the major oxygen-insensitive nitroreductase, NfsA7 (Table 1), although it reduces many of the same substrates. NTR is highly homologous to the “classical” nitroreductases of Salmonella typhimurium8 and Enterobacter cloacae.9 It also shows a lower degree of sequence homology to FRase I, the major flavin reductase of Vibrio fischeri.10 The FRase/NTR family of proteins also shares weak homology to a NADH oxidase (NOX) from Thermus thermophilus.11
The substrate specificities of the FRases and nitroreductases differ, perhaps reflecting divergent physiological roles. Whereas the FRases of Vibrio species provide reduced FMN for bioluminescence reactions,12 NTR cannot reduce free flavin molecules. The physiological role of nitroreductases is unclear; it may be to metabolise certain xenobiotics. The nitroreductase of S. typhimurium is a major contributor to the reductive reactions of the Ames mutagenicity test;13 those from E. cloacae and several other species have been shown to reduce TNT, and there is interest in their use for bioremediation.14, 15
E. coli NTR is of current interest for use in the gene therapy of cancer,16 based on its ability to generate cytotoxic products by reduction of the prodrug CB1954.17 Expression of NTR in human tumour cells has been shown to increase their sensitivity to CB1954 by up to 2500-fold, and treatment of mice with CB1954 can lead to significant growth delay or regression of tumours that express NTR.18, 19 However, CB1954 is a relatively poor substrate for NTR, due to both a high Km and a low kcat value,3 which may limit the therapeutic potential of the wild-type enzyme. In order to enhance the possible clinical efficacy of NTR and CB1954 for cancer gene therapy, we aim to engineer NTR for improved activity with the prodrug. A complementary approach aims to develop alternative prodrugs that would be activated more efficiently by NTR.5 To provide the basis for rational design, both of site-directed mutants and of alternative prodrugs, we have determined the crystal structure of NTR complexed with a substrate analogue. This paper reports three high-resolution X-ray structures of E. coli NTR complexed with nicotinic acid (NIC-NTR), from tetragonal, monoclinic and orthorhombic crystal forms (at 1.7 Å, 1.8 Å and 2.4 Å resolution, respectively), yielding ten non-crystallographically related monomers. Comparisons of the ten monomers allows greater confidence in the structure, particularly the side-chain conformations.
Section snippets
Description of the fold
Three crystal forms of the NTR-nicotinic acid complex were obtained under the same crystallization conditions. Native protein gave both tetragonal and monoclinic crystals, whilst selenomethionyl protein gave both tetragonal and orthorhombic crystals. The structure of the orthorhombic form was initially determined by multiwavelength anomalous dispersion (MAD); the tetragonal form was then solved by single wavelength anomalous dispersion (SAD); the monoclinic form was solved by molecular
Analysis of the three crystal forms
Three different crystal forms of the NIC-NTR complex were obtained using the same crystallization conditions. The analysis of all ten monomers from these allows the effects of crystal contacts to be evaluated. Both native and selenomethionyl protein produced tetragonal crystals with the same cell dimensions (within 1%); however, monoclinic crystals were only obtained with native protein while orthorhombic crystals were only obtained with selenomethionyl protein (Table 2). The monoclinic and
Protein preparation and crystallization
The nitroreductase gene was amplified by PCR from E. coli DH5α, and cloned into a derivative of pET11c. Its sequence was found to be identical to that previously reported.3, 30 The plasmid was introduced into E. coli BL21 cells, and expression of nitroreductase induced using 1 mM IPTG. Nitroreductase was purified as described.3 An additional step, involving passage of the protein through a hydroxyapatite matrix (in 10 mM sodium phosphate buffer, pH 7.0) was found to remove minor impurities and
Acknowledgements
We thank Dr Jane Grove (Cobra Therapeutics and University of Birmingham, UK) for the nitroreductase clone; Dr Gill Anlezark (CAMR, Porton Down, UK) for helpful discussions and advice on NTR purification; Dr Klaus Fütterer (University of Birmingham, UK) for fruitful discussion; Drs Raimond Ravelli, Sean McSweeney and Hassan Belrhali (ESRF, Grenoble, France) for assistance with data collection; and Drs Kornelia Jumel and Steve Harding (National Centre for Hydrodynamic Studies, University of
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