title: Purification and characterization of NAD(P)H-dependent nitroreductase I from Klebsiella sp. C1 and enzymatic transformation of 2,4,6-trinitrotoluene.
abstract: Three NAD(P)H-dependent nitroreductases that can transform 2,4,6-trinitrotoluene (TNT) by two reduction pathways were detected in Klebsiella sp. C1. Among these enzymes, the protein with the highest reduction activity of TNT (nitroreductase I) was purified to homogeneity using ion-exchange, hydrophobic interaction, and size exclusion chromatographies. Nitroreductase I has a molecular mass of 27 kDa as determined by SDS-PAGE, and exhibits a broad pH optimum between 5.5 and 6.5, with a temperature optimum of 30-40 degrees C. Flavin mononucleotide is most likely the natural flavin cofactor of this enzyme. The N-terminal amino acid sequence of this enzyme does not show a high degree of sequence similarity with nitroreductases from other enteric bacteria. This enzyme catalyzed the two-electron reduction of several nitroaromatic compounds with very high specific activities of NADPH oxidation. In the enzymatic transformation of TNT, 2-amino-4,6-dinitrotoluene and 2,2',6,6'-tetranitro-4,4'-azoxytoluene were detected as transformation products. Although this bacterium utilizes the direct ring reduction and subsequent denitration pathway together with a nitro group reduction pathway, metabolites in direct ring reduction of TNT could not easily be detected. Unlike other nitroreductases, nitroreductase I was able to transform hydroxylaminodinitrotoluenes (HADNT) into aminodinitrotoluenes (ADNT), and could reduce ortho isomers (2-HADNT and 2-ADNT) more easily than their para isomers (4-HADNT and 4-ADNT). Only the nitro group in the ortho position of 2,4-DNT was reduced to produce 2-hydroxylamino-4-nitrotoluene by nitroreductase I; the nitro group in the para position was not reduced.
authors.affiliation: Division of Biological Sciences, Kangwon National University, Hyoja-dong 192-1, Chuncheon, 200-701, South Korea.
authors.forename: Hyoun-Young
authors.initials: HY
authors.lastname: Kim
authors.forename: Hong-Gyu
authors.initials: HG
authors.lastname: Song
mesh_terms: D000595:Amino Acid Sequence; D007709:Klebsiella / Q000201:enzymology*; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism*; D009601:Nitroreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010084:Oxidation-Reduction; D014303:Trinitrotoluene / Q000378:metabolism*
chemical_list: D014303:Trinitrotoluene; D009249:NADP; D009601:Nitroreductases