The 3D‐structure, kinetics and dynamics of the E. coli nitroreductase NfsA with NADP⁺ provide glimpses of its catalytic mechanism

Abstract

Nitroreductases activate nitroaromatic antibiotics and cancer prodrugs to cytotoxic hydroxylamines and reduce quinones to quinols. Using steady-state and stopped-flow kinetics, we show that the Escherichia coli nitroreductase NfsA is 20-50 fold more active with NADPH than with NADH and that product release may be rate-limiting. The crystal structure of NfsA with NADP + shows that a mobile loop forms a phosphate-binding pocket. The nicotinamide ring and nicotinamide ribose are mobile, as confirmed in molecular dynamics (MD) simulations. We present a model of NADPH bound to NfsA. Only one NADP + is seen bound to the NfsA dimers, and MD simulations show that binding of a second NADP(H) cofactor is unfavourable, suggesting that NfsA and other members of this protein superfamily may have a half-of-sites mechanism.

Structure and Dynamics of Three Escherichia coli NfsB Nitro-Reductase Mutants Selected for Enhanced Activity with the Cancer Prodrug CB1954

“…Despite the lower dissociation constants of the mutants for CB1954 than that of the wild type, we were unable to obtain crystals of the proteins with bound CB1954 or related dintrobenzamide prodrugs, even at neutral pH where the prodrugs are stable. Given that the binding of nitroaromatic antibiotics is reversed in oxidized and reduced NfsB [ 30 ] and NfsA [ 40 ], it is possible that the prodrugs only bind to the reduced enzyme or with NAD(P) + in one of the two active sites [ 39 , 46 ]. Future work will concentrate on modelling the interactions with the reduced enzymes.…”

Oxygen‐insensitive nitroreductase E. coliNfsA, but not NfsB, is inhibited by fumarate

“…In both enzymes the substrates interact with a hydroxyl group at position 41´, Ser 41´in NfsA, and Thr 41´in NfsB. In NfsB, substrates interact with Phe 124´in the same subunit as Thr 41´2 6,27 whereas in NfsA substrates interact with Arg 225 of the other subunit, 22,23 neither of which has a counterpart in the other enzyme (Figure 5A,B). The absence of the charged residues Arg 225 and Arg 133 in NfsB, means that it does not bind fumarate.…”

“…The structure of E. coli NfsA has been determined in the absence of substrates by Kobori et al (1F5V). 21 Recently, we determined the structure of NfsA from E. coli in complex with the substrates nitrofurantoin, quinone, with the product hydroquinone and with an inhibitor bound FMN 22 and, separately, with the cofactor NADP + 23 . We and others have also determined the structure of free NfsB 24 , 25 and of NfsB in complex with nicotinic acid 26 and nitrofurazone.…”

Synthesis of Pharmaceutically Relevant Arylamines Enabled by a Nitroreductase from Bacillus tequilensis

“…[20,22] This is because substrates tend to form aromatic stacking interactions with the flavin cofactor, as indicated by crystal structures and molecular dynamic studies. [23][24][25] The extent of these interactions depends on the size of the substrate's π system. Therefore, the ability to perform the full nitro reduction to the respective amine depends not only on the prosthetic flavin's redox potential but also on many other factors.…”

“…Additionally, the presence of large π systems in a substrate also determines its likelihood of being reduced by the enzyme [20,22] . This is because substrates tend to form aromatic stacking interactions with the flavin cofactor, as indicated by crystal structures and molecular dynamic studies [23–25] . The extent of these interactions depends on the size of the substrate's π system.…”