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. 2011:1:63.
doi: 10.1038/srep00063. Epub 2011 Aug 12.

Activation of nitrofurazone by azoreductases: multiple activities in one enzyme

Ali Ryan  1 , Elise Kaplan, Nicola Laurieri, Edward Lowe, Edith Sim
Affiliations

Activation of nitrofurazone by azoreductases: multiple activities in one enzyme

Ali Ryan et al. Sci Rep. 2011.

Abstract

Azoreductases are well known for azo pro-drug activation by gut flora. We show that azoreductases have a wider role in drug metabolism than previously thought as they can also reduce and hence activate nitrofurazone. Nitrofurazone, a nitroaromatic drug, is a broad spectrum antibiotic which has until now been considered as activated in bacteria by nitroreductases. The structure of the azoreductase with nitrofurazone bound was solved at 2.08 Ã… and shows nitrofurazone in an active conformation. Based on the structural information, the kinetics and stoichiometry of nitrofurazone reduction by azoreductase from P. aeruginosa, we propose a mechanism of activation which accounts for the ability of azoreductases to reduce both azo and nitroaromatic drugs. This mode of activation can explain the cytotoxic side-effects of nitrofurazone through human azoreductase homologues.

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Figures

Figure 1
Figure 1. Structure of nitrofurazone.
Figure showing the two nitrofurazone tautomers that occur in equilibrium in solution.
Figure 2
Figure 2. Binding of nitrofurazone to paAzoR1.
A) The binding of nitrofurazone in its two alternate conformers are shown with brown carbon atoms. The FMN is shown with yellow carbon atoms, both conformations of nitrofurazone are shown with brown carbon atoms. Important side chains are labelled. Blue mesh is the refined 2Fo-Fc map contoured at 1σ, while black mesh is unbiased Fo-Fc map at 2.2σ. The black sphere represents a bound water molecule. (B) Comparison of balsalazide and nitrofurazone binding to paAzoR1. Colouring of nitrofurazone is as in (A) and balsalazide is in purple. This figure was produced in Pymol v1.1.
Figure 3
Figure 3. Comparison of the overall structures (A–C) and nitroaromatic binding (D–F) of paAzoR1 (A,D), hNQO2 (B,E) and NfsB (C,F).
(A–C) The overall structure of the dimeric forms of each of the three proteins. In all three panels monomer A is in green and B is in turquoise. (D–F) A detailed view of nitroaromatic drug binding within the active site of the enzymes (nitrofurazone in paAzoR1 and NfsB and CB1954 in hNQO2). In all panels the flavin cofactor is in yellow (FMN for paAzoR1 and NfsB, FAD for hNQO2). The Nitroaromatic drug is in brown in panels D–F. The structure of hNQO2 bound to CB1954 is from PDB 1XI2 and the structure of NfsB bound to nitrofurazone is from PDB 1YKI.
Figure 4
Figure 4. Mechanism for nitrofurazone reduction.
(A) First round of nitrofurazone reduction. (B) Second round of nitrofurazone reduction resulting in hydroxylamine formation. The proton in both (A) and (B) is likely to be donated by a stably bound water molecule observed in the structure (Fig. 2A). Before (A) and between (A) and (B) NADPH binds to paAzoR1 and transfers a hydride to FMN before being replaced by nitrofurazone.
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References

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