In silico structure–function analysis of E. cloacae nitroreductase

Abstract

Reduction, catalyzed by the bacterial nitroreductases, is the quintessential first step in the biodegradation of a variety of nitroaromatic compounds from contaminated waters and soil. The Enterobacter cloacae nitroreductase (EcNR) enzyme is considered as a prospective biotechnological tool for bioremediation of hazardous nitroaromatic compounds. Using diverse computational methods, we obtain insights into the structural basis of activity and mechanism of its function. We have performed molecular dynamics simulation of EcNR in three different states (free EcNR in oxidized form, fully reduced EcNR with benzoate inhibitor and fully reduced EcNR with nitrobenzene) in explicit solvent and with full electrostatics. Principal Component Analysis (PCA) of the variance-covariance matrix showed that the complexed nitroreductase becomes more flexible overall upon complexation, particularly helix H6, in the vicinity of the binding site. A multiple sequence alignment was also constructed in order to examine positional constraints on substitution in EcNR. Five regions which are highly conserved within the flavin mononucleotide (FMN) binding site were identified. Obtained results and their implications for EcNR functioning are discussed, and new plausible mechanism has been proposed.

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

“…Substrate reduction by reduced flavin commonly occurs via hydride transfer (Fagan and Palfey, 2010) in which two electrons are transferred as an H − . Yet in NR’s case H equivalents have been proposed to be transferred as solvent-derived protons via electron-coupled proton transfer (Christofferson and Wilkie, 2009; Isayev et al, 2012). …”

“…This has been proposed to occur via serial single electron and proton transfers (Fig. 1B, (Christofferson and Wilkie, 2009; Isayev et al, 2012)) based on the distances involved. Overall, the structures of NR and homologues complexed with oxidizing substrates reveal distances from the flavin N5 to the oxidizing moiety varying from 3.8 – 7.7 Å (Haynes et al, 2002; Hecht et al, 1995; Kobori et al, 2001; Koike et al, 1998; Parkinson et al, 2000; Race et al, 2007; Tanner et al, 1996).…”

“…1B) (Page, 1999). Indeed, molecular dynamics (MD) simulations of NfsB and NR found that the substrates CB1954 and nitrobenzene did not remain bound in orientations placing the nitro group near N5, but reoriented or dissociated (Christofferson and Wilkie, 2009; Isayev et al, 2012). Therefore water H-bonded to the ~NOO was proposed to donate protons in conjunction with electron transfer from flavin.…”

Monoterpenes as nitrofurantoin resistance modulating agents: minimal structural requirements, molecular dynamics simulations, and the effect of piperitone on the emergence of nitrofurantoin resistance in Enterobacteriaceae

“…After a short decrease in RMSD at 7500 ps, this value increased to 0.22 nm at 14,000 ps and up to 20,000 ps, which indicates that the system reached equilibrium. This variation in RMSD can be attributed to the breathing motion of enzymes [31,32]. The flexibility of NR results in broad substrate specificity and the NR-related enzymes can reduce a variety of nitroaromatic compounds [33][34][35][36].…”

“…With regard to the experimental X-ray structure of NR, the high-temperature factor of NR indicates high mobility and plasticity [32]. The Thr 110, with a backbone RMSF value of 0.3 nm, is situated in H6.…”