The structure of Escherichia coli nitroreductase complexed with nicotinic acid: three crystal forms at 1.7 A, 1.8 A and 2.4 A resolution.

Escherichia coli nitroreductase is a flavoprotein that reduces a variety of quinone and nitroaromatic substrates. Its ability to convert relatively non-toxic prodrugs such as CB1954 (5-[aziridin-1-yl]-2,4-dinitrobenzamide) into highly cytotoxic derivatives has led to interest in its potential for cancer gene therapy. We have determined the structure of the enzyme bound to a substrate analogue, nicotinic acid, from three crystal forms at resolutions of 1.7 A, 1.8 A and 2.4 A, representing ten non-crystallographically related monomers. The enzyme is dimeric, and has a large hydrophobic core; each half of the molecule consists of a five-stranded beta-sheet surrounded by alpha-helices. Helices F and F protrude from the core region of each monomer. There is an extensive dimer interface, and the 15 C-terminal residues extend around the opposing monomer, contributing the fifth beta-strand. The active sites lie on opposite sides of the molecule, in solvent-exposed clefts at the dimer interface. The FMN forms hydrogen bonds to one monomer and hydrophobic contacts to both; its si face is buried. The nicotinic acid stacks between the re face of the FMN and Phe124 in helix F, with only one hydrogen bond to the protein. If the nicotinamide ring of the coenzyme NAD(P)H were in the same position as that of the nicotinic acid ligand, its C4 atom would be optimally positioned for direct hydride transfer to flavin N5. Comparison of the structure with unliganded flavin reductase and NTR suggests reduced mobility of helices E and F upon ligand binding. Analysis of the structure explains the broad substrate specificity of the enzyme, and provides the basis for rational design of novel prodrugs and for site-directed mutagenesis for improved enzyme activity.