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

Abstract Escherichia coli NfsA and NfsB are founding members of two flavoprotein families that catalyze the oxygen‐insensitive reduction of nitroaromatics and quinones by NAD(P)H. This reduction is required for the activity of nitrofuran antibiotics and the enzymes have also been proposed for use with nitroaromatic prodrugs in cancer gene therapy and biocatalysis, but the roles of the proteins in vivo in bacteria are not known. NfsA is NADPH‐specific whereas NfsB can also use NADH. The crystal structures of E. coli NfsA and NfsB and several analogs have been determined previously. In our crystal trials, we unexpectedly observed NfsA bound to fumarate. We here present the X‐ray structure of the E. coli NfsA‐fumarate complex and show that fumarate acts as a weak inhibitor of NfsA but not of NfsB. The structural basis of this differential inhibition is conserved in the two protein families and occurs at fumarate concentrations found in vivo, so impacting the efficacy of these proteins.

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. 27 Several structures of NfsA and NfsB homologs 28,29 and mutants have also been determined. In our initial attempts to crystallize NfsA, we unexpectedly found a four-carbon, dicarboxylic acid in the active site. In this work, we show the structure of the NfsA complex with fumarate and kinetic inhibition assays with both NfsA and NfsB. NfsA but not NfsB is inhibited at in vivo concentrations of fumarate, but only much higher concentrations of succinate. This inhibition could be significant for future use in biosynthesis and therapeutic applications and may help indicate the in vivo role of NfsA.

| Protein expression and purification
E. coli NfsA was overexpressed in E. coli BL21 (λDE3) without any tags, from the pET-24 derivative pPS1341A1, encoding NfsA under the control of a T7 promoter, as described in Vas et al. 14 It was purified as described previously, using ammonium sulfate precipitation, hydrophobic interaction chromatography on phenyl sepharose, ion exchange chromatography on Q sepharose, followed by size exclusion chromatography on Sephacryl 200 or Superdex 75. E. coli NfsB was over expressed from a pET-11c plasmid derivative and purified using similar methods. 26 Protein concentrations were estimated by Bradford assay 30  Data were collected on a Rigaku 007HF generator with a Saturn CCD detector mounted on a 4-circle kappa goniometer.
Diffraction images were indexed, integrated, and processed using MOSFLM, 32 iMOSFLM, 33 or XDS. 34 Datasets were combined and scaled using POINTLESS and SCALA 35 and data quality was assessed using XTRIAGE. 36 All structures were solved by molecular replacement with PHASER, 37 using the published NfsA structure PDB entry 1F5V for NfsA 21 as the starting model. Structures were refined using REFMAC5 38 and then with PDB-Redo. 39 Models were built and modified using Coot. 40 Final models were validated using MOLPROBITY 41 and POLYGON. 42 The structural figures were drawn using UCSF Chimera 1.13.1. 43

| Steady state enzyme assays
Steady-state kinetic assays were monitored spectrophotometrically, over 1-2 min, as described previously 27 Experiments were performed in 10 mM Tris-HCl pH 7.0, at 25 C, at 50 mM total ionic strength for fumarate, assuming that is doubly charged at this pH. Succinate where  F I G U R E 1 X-ray structure of fumarate bound to NfsA. (A) Ribbon diagram of NfsA dimer, with bound fumarate. One subunit is in gray and the other is in rainbow colors blue to red from N-to C-terminus. The FMN cofactor is shown as ball and stick, with C atoms in yellow, N blue, oxygen red, and phosphorus orange. The side chains that interact with the bound fumarate are shown as sticks, with carbon atoms colored as the ribbon backbone, and heteroatoms as for FMN. The ligand is shown in tan, with oxygen atoms in red. (B,C) Two orientations of fumarate bound to NfsA. The FMN and fumarate are shown in ball and stick, colored as in A. The side chains that interact with fumarate are shown as sticks, labeled and colored as in A, with Ser 41, from the opposite subunit to the other interacting residues, given a prime notation. Cyan lines show the hydrogen bonding to the ligand. The mesh shows the electron density within a radius of 2 Å from the fumarate (level 0.53 e) at 1 sigma.
PEG. Crystallization of the protein in the new PEG in the presence of fumarate allowed us to confirm the structure and interactions of the acid and the protein ( Table 1).
The structure of the protein in the presence of fumarate is fully symmetrical, with only one subunit in the asymmetric unit, and shows the characteristic dimeric, alpha/beta fold of NfsA ( Figure 1A). Each monomer has a core domain of four beta strands surrounded by alpha

| Steady-state kinetics with NfsA
Steady-state kinetics studies showed that neither succinate nor fumarate are substrates for NfsA but both act as inhibitors of NfsA, largely competitive with respect to NADPH. In order to ensure that the effects were not due to changes in ionic strength, the reactions were done at the same ionic strength with and without an inhibitor. At 50 mM ionic strength, fumarate gave an inhibition constant of 145 ± 28 μM with respect to NADPH and, six-fold weaker, 960 ± 390 μM, inhibition with respect to nitrofurazone (Figure 2A,B, Table 2). Succinate showed much weaker inhibition than fumarate, so reactions were repeated with higher concentrations of succinate, up to 30 mM. The reactions with succinate were therefore done at higher total ionic strength, 150 mM. These showed that succinate has a K i of 4.3 ± 0.6 mM with respect to NADPH, but no inhibition with respect to nitrofurazone was detected ( Figure 3A,B, Table 2

| No effect with NfsB
Kinetic assays of NfsB showed no effect of either fumarate or succinate at 1 mM concentration (Figure 4, Table S1). Comparison of the structure of NfsB 26 with that of NfsA ( Figure 5) shows that, despite limited sequence homology, they have a similar core structure of $180 amino acids containing five helices and four beta strands, intertwined in a stable dimer. The FMN cofactor packs on the core of the protein, interacting with both subunits, and has similar interactions in NfsA and NfsB. However, while the known range of substrates for both enzymes are similar and these stack upon the FMN ring in a similar way, they interact differently with the two proteins. NfsB contains a 2-helix insertion (residues 95-132, colored gold and magenta for the two different subunits in Figure 5) before the long, central helix of  the core structure, with one helix stacking against the side of the substrate. In contrast, NfsA has an approximately 60 aa C-terminal extension (residues 180-240), after the chain has crossed the dimer interface. 9 This region forms the phosphate-binding pocket for the NADPH cofactor and then extends back across the dimer interface, over the substrate, so that a different subunit is over the substrate from that in NfsB. The extensions make the shape of the active site, the channels into it, and the interactions with the substrates very different between the two enzymes. 44 The active site cavity of NfsB is smaller and much less positively charged than that of NfsA, ( Figure 5C,D). 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.

| In vivo effect of fumarate
While the dissociation constant for fumarate from NfsA is high, as shown in the Ki measurements above, it is within the cellular concentration in E. coli ($0.1 mM cf succinate 0.5 mM) 45  as is the upstream gene in the operon, ybjC, which is thought to be a membrane-bound oxidase. NfsB, which also reduces quinones, has a higher K m for most substrates (for example K m NFZ of NfsB in Table S1 cf K m NFZ of NfsA in Table 2) and so repression of this enzyme may be less important.

CONFLICT OF INTEREST
No commercial interest.
F I G U R E 5 Comparison of the X-ray structures of NfsB and NfsA.
(A) Ribbon diagram of NfsB dimer with bound nicotinate, from 1ICR, 26 in the same orientation as NfsA in Figure 1. The core residues of the two subunits are in gray and blue, with residues 95-132, not found in NfsA, in in gold and magenta, respectively. The FMN cofactor is shown as ball and stick, with C atoms in yellow, N blue, O red, and P orange. The side chains that interact with the bound nicotinate are labeled and shown as sticks, with carbon atoms colored as the ribbon backbone, and heteroatoms as for FMN. The nicotinate ligand is shown in ball and stick with C atoms in gray and others in CPK colors. (B) Ribbon diagram of NfsA dimer with bound fumarate, in the same orientation as NfsB. One subunit is in tan and the other in cyan, with residues 180-240, not found in NfsB, in gold and magenta, respectively. The side chains that interact with the bound fumarate are labeled and shown as sticks, with carbon atoms colored as the ribbon backbone, and heteroatoms as for FMN. The FMN is colored as in A with the fumarate in ball and stick with C atoms in gray and oxygen in red. (C,D) Coulombic surface representation of NfsB, and NfsA respectively, in the same orientation as in A and B. The FMN and ligands are shown as ball and stick, colored as in A and B. The surface is colored red through white to blue corresponding to negative, through uncharged to positive charge.

PEER REVIEW
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DATA AVAILABILITY STATEMENT
The crystallographic data has been deposited in the Protein Data Bank with accession code 8AJX.