Characterization Of Nitroreductases From Escherichia Coli In Controllable Synthesis Of Arylhydroxylamines

Arylhyroxylamines are important intermediates having been widely used in synthesis of pesticides, pharmaceuticals, and fine chemicals. Biocatalysis, especially enzymes reduction offer a green, high selective and controllable method for the synthesis of arylhydroxylamines. Here we use E.coli nitroreductases NfsA and NfsB to synthesize arylhydroxylamines. NfsA and NfsB are NAD(P)H-dependent flavoproteins, can catalyze the reduction of nitro groups to hydroxylamino products with high regioselectivity. The aim of this paper was to study the characterizations of NfsA and NfsB in controllable synthesis of arylhydroxylamines. This paper carried out the following tasks:(1) The purified recombinant NfsA, NfsB and NfsB mutant F123A were obtained and essayed. Enzyme-activity assay, HPLC and MS analyses indicated that NfsA and NfsB both presented catalytic activity toward nitro substrate 1,2-DNB,1,3-DNB and 1,4-DNB, with hydroxylaminos as their final products. The kinetic experiments showed that the kcat/Km values of NfsB for 1,2-DNB,1,3-DNB and 1,4-DNB were 13%,33% and 50% of NfsA, respectively. The order of catalytic efficiency of three dinitrobenzenes is 1,2-DNB>1,3-DNB>1,4-DNB. Docking experiments indicated that 1,2-DNB could be exactly positioned in the enzyme active pocket due to the relative location of two nitro groups. In addition, NfsB mutant F123A significantly improved catalytic efficiency toward dinitrobenzenes, the kcat/Km values were 2-5 fold of wild type NfsB accordingly. It suggested that Phel23 was an important residue affecting enzyme activity, may exert steric effects on substrate binding.(2) To investigate the effects of substituent groups (-NH2,-COCl-COOH,-CH3) on substrate reactivity, substituted dinitrobenzenes (3,5-DNA,3,5-DNT, DNBC and DNBA) were studied. Enzyme activity assay, HPLC and MS analyses indicated that NfsA and NfsB both presented catalytic activity toward four substituted dinitrobenzenes, with hydroxylaminos as their final products. The kinetic experiments showed that the kcat/Km values of NfsA and NfsB for DNBC and DNBA were 3-17 fold of 1,3-DNB, while the kcat/Km values for 3,5-DNA and 3,5-DNT were 63%-72%of 1,3-DNB, suggesting that the characterizations of the substituted groups may affect dinitrobenzene reactivity. NfsB mutant F123A significantly improved catalytic efficiency for substituted dinitrobenzenes.(3) Based on sequence alignment and structural analysis of NfsA and NfsB, we presumed that the amino acid residues Argl5, Asp165, Lys167, Tyr199, Tyr200 and Arg225 in NfsA, Lys14, Thr41, Asn71, Phe123 and Phel24 in NfsB may play critical roles in coenzyme binding. Site-directed mutants were designed to change coenzyme preference. The nitroreductase activities of NfsA, NfsB and their mutants with NADPH and NADH as coenzymes were compared, showing that the coenzyme preference of NfsA variants R15E, R225H and R225Q was converted into NADPH preference, the coenzyme preference of D165R and Y200A was converted into NADH preference, suggesting that Argl5, Aspl65, Tyr200 and Arg225 may affect the binding of cofactor and enzyme. Nitroreductase activities of NfsB variants K14R, K14S, N71S and N71T were significantly decreased using either NADPH or NADH as cofactors, while their coenzyme preference was converted into NADH preference, suggesting that Lys14, Asn71 may contribute to coenzyme binding.