Expression, Purification And Characterization Of NfsA From Escherichia Coli

Bacterial nitroreductases are homodimeric flavoproteins that can catalyze the NADPH-dependent reduction of nitro groups. Nitroreductases have raised great interests due to their potential applications in bio re mediation, biocatalysis, and bio medicine. Three nitroreductases, major oxygen-insensitive nitroreductases Nfs A, minor oxygen-insensitive nitroreductases Nfe B and YdjA, have been identified in E. coli. NfsA and NfsB belong to two different nitroreductase groups, Group A and Group B, and share low similarity on the amino acid sequence but higher biochemical and structural features. NfsA is less studied. In order to investigate the catalytic properties of NfsA, we carried out the following tasks:(1) NfsA encoding gene was amplified from E. coli K12strain using a pair of PCR primers designed on the basis of reference sequence (GenBank:BAA07425.1). The nfsA gene then was cloned to pET28a vector, and transformed into E. coli BL21(DE3). Expression conditions were optimized to obtain a high expression of the recombinase. Affinity chromatography was used to obtain a purified enzyme, which was verified by SDS-PAGE and western blot.(2) The structural comparison revealed the residue Phe42was present in the active site of NfsA, but was absent in NfsB. To investigate the role of the Phe42, variants F42Y, F42N and F42A were generated. Nitroreductase activity assay indicated that three variants lost activity toward the substrate nitrofurazone (NFZ) by a percentage of51.7%,96.3%, and98.7%, respectively. The kinetic parameters of NfsA and its variants for nitroaromatic substrates including5-(aziridin-1-yl)-2,4-dinitrobenzamide(CB1954),2,4,6-Trinitrotoluene (TNT),2,4-dinitrotoluene (2,4-DNT), and nitrofurazone(NFZ) were measured. Compared with the wild type, the variants did not significantly alter the Km values, but dramatically decreased kcat values and thus the ratio of kcat/Km. Far-UV CD spectra comparison of the wild type and F42Y suggested that there were no significant conformational changes occurred. However, F42A and F42N showed distinct changes at the range of the wavelength208nm to222nm, which can be attributed to the loss of the helix content. These findings revealed that Phe42in NfsA is important in maintaining enzyme activity and structure architecture.(3) Structural based sequence alignment of Group A and Group B nitroreductases refers to the amino acid residues Arg15, Lys167and Arg203may play critical roles in NADPH binding. Variants R15A, K167A, R203A and R203D were thus generated by site-directed mutagenesis. Nitroreductase activities and the kinetic parameters of NfsA and its variants using NADH and NADPH as cofactor were measured. The nitroreductase activities of four variants dependant NADPH are significantly decreased compared with the wild type, which indicates that these residules may contribute to co factor binding. K167A improved the activity when it used NADH as co factor, suggesting that Lys167may affect the coenzyme preference of the enzyme.In summary, nitroreductase NfsA was cloned and expressed. Critical amino acid resiudes contributed to its catalytic activity, coenzyme binding and cofactor preference were determined.