Biochemical characterization of trinitrotoluene transforming oxygen-insensitive nitroreductases from Clostridium acetobutylicum ATCC 824.
The genes that encode oxygen-insensitive nitroreductases from Clostridium acetobutylicum possessing 2,4,6-Trinitrotoluene (TNT) transformation activity were cloned, sequenced and characterized. The gene products NitA (MW 31 kDa) and NitB (MW 23 kDa) were purified to homogeneity. The NitA and NitB are oxygen-insensitive nitroreductases comprised of a single nitroreductase domain. NitA and NitB enzymes show spectral characteristics similar to flavoproteins. The biochemical characteristics of NitA and NitB are highly similar to those of NfsA, the major nitroreductase from E. coli. NitA exhibited broad specificity similar to that of E. coli NfsA and displayed no flavin reductase activity. NitB showed broad substrate specificity toward nitrocompounds in a pattern similar to NfsA and NfsB of Escherichia coli. NitB has high sequence similarity to NAD(P)H nitroreductase from Archaeoglobus fulgidus. NitA could utilize only NADH as an electron donor, whereas NitB utilized both NADH and NADPH as electron donors with a preference for NADH. The activity of both nitroreductases was high toward 2,4-Dinitrotoluene (2,4-DNT) as a substrate. Both the nitroreductases were inhibited by dicoumarol and salicyl hydroxamate. The nitroreductases showed higher relative expression on induction with TNT, nitrofurazone and nitrofurantoin compared to the uninduced control.
Kutty
Razia
R
Department of Biochemistry and Cell Biology MS-140, Rice University, Houston, TX 77005-1892, USA.
Bennett
George N
GN
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D046969:Clostridium acetobutylicum / Q000201:enzymology* / Q000235:genetics; D001728:Dicumarol / Q000494:pharmacology; D004136:Dinitrobenzenes / Q000378:metabolism; D019008:Drug Resistance, Neoplasm; D004791:Enzyme Inhibitors / Q000494:pharmacology; D029968:Escherichia coli Proteins / Q000378:metabolism; D005420:Flavoproteins / Q000737:chemistry; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009574:Nitro Compounds / Q000378:metabolism; D009582:Nitrofurantoin / Q000494:pharmacology; D009583:Nitrofurazone / Q000494:pharmacology; D009601:Nitroreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012457:Salicylamides / Q000494:pharmacology; D013057:Spectrum Analysis; D013379:Substrate Specificity; D014303:Trinitrotoluene / Q000378:metabolism*
D004136:Dinitrobenzenes; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D005420:Flavoproteins; D009574:Nitro Compounds; D011994:Recombinant Proteins; D012457:Salicylamides; D009243:NAD; D014303:Trinitrotoluene; D009249:NADP; C016403:2,4-dinitrotoluene; D001728:Dicumarol; C005703:salicylhydroxamic acid; D009582:Nitrofurantoin; C116053:NfsA protein, E coli; D009601:Nitroreductases; D009583:Nitrofurazone