Molecular Engineering And Its Catalytic Characteristics Of Escherichia Coli Nitroreductase NfsB

Nitroreductases are widely distributed flavoenzymes that require a prosthetic group FMN or FAD to catalyze the NAD(P)H-dependent reduction of nitro groups to corresponding hydroxylamino groups and/or amino groups. Thus, they have great potentials in bioremediation, biosynthesis of arylhydroxylamines, and prodrug activation for cancer treatments.This study focused on the catalytic features of the nitroreductase NfsB from Escherichia coli, using the widespread environmental contaminant 2,4,6-trinitrotoluene (TNT), the raw material for arylhydroxylamines 2,4-dinitrotoluene (2,4-DNT), and the cancer-treatment prodrug CB1954 as substrates. The reduced products were analyzed by HPLC and MS, and the catalysis kinetic parameters were then determined. Through a combination of site-directed mutagenesis, structural analysis, and molecular docking, the structural basis of the catalytic activity and regioselectivity of NfsB were proposed. Finally, a gene-engineered NfsB was obtained with high catalytic activity and regioselectivity.Main results obtained by this work were summarized below:(1) NfsA and NfsB have similar activities toward the substrate TNT. HPLC-MS analysis of reduced products showed that the transformation pathway of TNT to 2,4-dihydroxylamino-6-nitrotoluene (2,4-DHANT) was identical. When oxygen was abundant, a new product 2,2’,6,6’ -tetranitro-4,4’-azoxytoluene (4,4’-azoxy) was formed by condensation of hydroxylamines and nitroso intermediates. Mutagenesis studies showed the mutation of the F123 had great effects on the catalytic activity. When compared to the wild-type NfsB, the mutant F123A exhibited a 1.4-fold increase in kcat/Km toward TNT, and a 3.6-fold increase and a 4.0-fold increase in kcat/Km toward its corresponding amine derivatives (2-ADNT and 4-ADNT), respectively.(2) The regioselectivity of NfsB toward 2,4-DNT was altered by gene mutagenesis. The wild-type NfsB selectively reduces the 4-NO2 group when 2,4-DNT is the substrate. To change the selectivity from 4-NO2 group to 2-NO2 group, four residues of NfsB T41, N71, F70, and F124, were chosen for mutagenesis. The triple mutant T41L/N71S/F124W showed high preference to reduce the 2-NO2 group of 2,4-DNT, and its crystal structure was obtained and resolved. The structural comparison between the wild-type and the mutant T41L/N71S/F124W indicated there was not any significant difference in overall structure. However, a few conformational changes were observed in the active pocket. The side chain of L41 was in a contact distance to the W124, forming a more hydrophobic region above FMN. Moreover, the rings of the side chains of both W124 and F70 directed to the opposite side of the pocket, generating a wider entrance of the active site.(3) Molecular docking analysis showed that the binding modes of 2,4-DNT were different in the wild-type NfsB and the mutant T41L/N71S/F124W. The wild-type NfsB allowed only one configuration of 2,4-DNT, in which the 4-NO2 group positioned in a pocket consisting of residues T41, E165, G166 and F124, and faced N5 of FMN. However, in the mutant T41L/N71S/F124W,2,4-DNT was positioned with the 1-methyl group bound in a hydrophobic region formed by the side chains of L41 and W124, and the 2-NO2 group adjacent to the 1-methyl group was close to the catalytic site.(4) To improve the catalytic activity and regioselectivity of NfsB toward CB1954, four residues T41, N71, F123, and F124, were site-or combined mutated. The single mutant F124W successfully reduced the 4-NO2 group instead of the 2-NO2 group of CB1954. Combination mutagenesis showed that N71S/F124W and F123A/F124W increased the catalytic activity, but had no effects on the regioselectivity. The triple mutant N71S/F123A/F124W regioselectively reduced the 4-NO2 group of CB1954, and exhibited a 22.1-fold increase in kcat/Km when compared to the wild-type.(5) The crystal structure of the mutant N71S/F123A/F124W was obtained and resolved. The structural comparison between the wild-type and the mutants N71S/F124W and N71S/F123A/F124W indicated that the larger indo ring of W124 might stack with the aziridinyl group of CB1954, faciliating the 4-NO2 group positioning above N5 of FMN. Substitution of F123 by A123 removed the steric constraint, and faciliated conformational changes of the adjacent residue F124. In the mutants N71S/F124W and N71S/F123A/F124W, the side chain of residue F70 adjacent to S71 was observed to be far away from the active pocket when compared to the wild-type, resulting in a wider entrance for substrate binding. Taken together, the triple mutation thus had achieved higher catalytic activity.