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

Abstract

Summary Nitroreductase from Enterobacter cloacae (NR) reduces diverse nitroaromatics including herbicides, explosives and prodrugs, and holds promise for bioremediation, prodrug activation and enzyme-assisted synthesis. We solved crystal structures of NR complexes with bound substrate or analog for each of its two half-reactions. We complemented these with kinetic isotope effect (KIE) measurements elucidating H-transfer steps essential to each half-reaction. KIEs indicate hydride transfer from NADH to the flavin consistent with our structure of NR with the NADH analog nicotinic acid adenine dinucleotide (NAAD). The KIE on reduction of p-nitrobenzoic acid (p-NBA) also indicates hydride transfer, and requires revision of prior computational mechanisms. Our mechanistic information provided a structural restraint for the orientation of bound substrate, placing the nitro group closer to the flavin N5 in the pocket that binds the amide of NADH. KIEs show that solvent provides a proton, enabling accommodation of different nitro group placements, consistent with NR’s broad repertoire.

Informing Efforts to Develop Nitroreductase for Amine Production

“…We compared the members of two different NR subgroups: St NfsB and Ms PnbA. The NfsB from S. typhimurium has been studied extensively and is a close relative of the NfsBs from E. cloacae and E. coli for which detailed mechanistic information is available [ 12 , 17 , 22 , 50 ]. The PnbA from M. smegmatis converts the nitroaromatic drug BTZ043 to the corresponding amine with a yield on the order of 30% [ 56 ].…”

“…To augment the substrate’s intrinsic propensity for reduction the enzyme must engage it in interactions that stabilize the transition state and position it for reaction by the reduced flavin (and a proton donor). However, recent studies on Ent NfsB demonstrate that substrate appears to bind in a poor geometry for nitroreduction [ 50 ]. Thus, a crucial second avenue for improvement is identification of active sites that provide geometry more conducive to reaction.…”

“…In particular, we propose that NfsB’s poor geometry for reduction of nitrogroups is unlikely to be general, and that another subgroup may provide a more suitable geometry for its reduction to amine. Moreover, given nitroaromatic binding in Ent NfsB treats the nitroaromatic like an analog of NADH, placing the nitrogroup where NADH’s amide binds, and the non-target aromatic ring near N5 where NADH’s hydride-bearing C4 binds [ 50 ], we propose that a subgroup that does not employ NADH as the reducing substrate may be the best choice of platform for aromatic nitro group reduction. The distinguishing structure of the different subgroups constrains the substrate-binding cavity on different sides ( Figure 5 a and Figure S7 ) consistent with the provenance of this structure from different locations in the amino acid sequence ( Figure 5 b [ 41 ]).…”

“…R(H) means Arg but sometimes His, R/K means Arg or Lys, P + 2 means that the second residue after a Pro contributes the interaction, S means Ser. The FMN from NfsB is included in yellow and an NADH analog bound to the NfsB model is in green, both with non-C atoms coloured by atom [ 50 ]; ( b ) Shows an alignment of consensus and representative sequences from each of these subgroups and HUB (brown label). The three insertions/extension in the sequence giving rise to the distinguishing structure are in boxes colored according to the subgroup in which it is best developed and labeled ‘E1’, ‘E2’ and ‘E3’ as per Akiva, Copp et al [ 41 ].…”

“…However, the shared core structure must also allow the different subgroups of the superfamily to promote different aspects of the flavin’s chemical repertoire. Thus it is anticipated that active site features shared within each subgroup interact with the flavin to modulate its activity in a common way [ 49 , 50 , 51 ], select a common category of substrate and/or position substrate in a way that is shared within the subgroup [ 52 , 53 , 54 , 55 ]. Conversely, given that some superfamily members have been reported to reduce nitroaromatics to corresponding amines, does this activity correlate with a particular subgroup, or active site features?…”