Structural and Mechanistic Studies of Escherichia coli Nitroreductase with the Antibiotic Nitrofurazone

Abstract

The antibiotics nitrofurazone and nitrofurantoin are used in the treatment of genitourinary infections and as topical antibacterial agents. Their action is dependent upon activation by bacterial nitroreductase flavoproteins, including the Escherichia coli nitroreductase (NTR). Here we show that the products of reduction of these antibiotics by NTR are the hydroxylamine derivatives. We show that the reduction of nitrosoaromatics is enzyme-catalyzed, with a specificity constant ϳ10,000-fold greater than that of the starting nitro compounds. This suggests that the reduction of nitro groups proceeds through two successive, enzyme-mediated reactions and explains why the nitroso intermediates are not observed. The global reaction rate for nitrofurazone determined in this study is over 10-fold higher than that previously reported, suggesting that the enzyme is much more active toward nitroaromatics than previously estimated. Surprisingly, in the crystal structure of the oxidized NTR-nitrofurazone complex, nitrofurazone is oriented with its amide group, rather than the nitro group to be reduced, positioned over the reactive N5 of the FMN cofactor. Free acetate, which acts as a competitive inhibitor with respect to NADH, binds in a similar orientation. We infer that the orientation of bound nitrofurazone depends upon the redox state of the enzyme. We propose that the charge distribution on the FMN rings, which alters upon reduction, is an important determinant of substrate binding and reactivity in flavoproteins with broad substrate specificity.

E. coli NfsA: an alternative nitroreductase for prodrug activation gene therapy in combination with CB1954

“…NfsB is a homodimeric flavoenzyme which can use either NADH or NADPH to reduce the tightly bound FMN; after dissociation of the NAD (P) þ ; a variety of nitroaromatic or quinone substrates can be reduced in a second reaction step Lovering et al, 2001;Race et al, 2005). The published K m for NfsB nitroreductase with CB1954 is 862 mM , which is considerably higher than its peak serum concentration achievable in humans (5 -10 mM) (Chung-Faye et al, 2001).…”

Identification of novel members of the bacterial azoreductase family in Pseudomonas aeruginosa

“…The structure of the active site of paMdaB (Fig 7B) is very similar to that of paAzoR1 (Fig 4A), it is a predominantly hydrophobic pocket surrounded by aromatic residues. In paMdaB Thr87 is in a similar position to Phe60 in paAzoR1 which has previously been identified to be important for determining substrate specificity of azoreductases [42]. In a model of paAzor3 it was suggested that the equivalent residue (Thr60), due to its strategic position at one of the entrances to the active site, would determine the accessibility to the active site for larger substrates [19].…”

Chalcones and N-acylhydrazones: direct analogues? Exploratory data analysis applied to potential novel antileishmanial agents

“…The crystallized structures of nitrofurazone and 2'-hydroxy-4''-dimethylamino-chalcone were retrieved from the Brookhaven Protein Data Bank (PDB entry code 1yki, resolution at 1.70 Å; Race et al, 2005) and file ob1067.cif (resolution at 1.01 Å; Liu et al, 2002), respectively, and were used as geometry references in the building up of all ligands. Crystallographic information of a nitrothiophene 2-carbaldehyde was retrieved from the file lh6379.cif (resolution at 1.18 Å; McBurney et al, 2005) and also used as a reference particularly in the construction of the nitrothiophene ring moiety.…”