Biochemical characterization of trinitrotoluene transforming oxygen-insensitive nitroreductases from <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> ATCC 824 |
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Authors: | Razia?Kutty Email author" target="_blank">George?N?BennettEmail author |
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Institution: | (1) Department of Biochemistry and Cell Biology MS-140, Rice University, Houston, TX 77005-1892, USA |
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Abstract: | 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. |
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Keywords: | Oxygen-insensitive nitroreductase Trinitrotoluene biotransformation Flavoprotein |
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