Characterization of 2,4-Dinitrotoluene Dioxygenase from Recombinant Esherichia coli Strain PFJS39: Its Direct Interaction with Vitreoscilla Hemoglobin

ABSTRACT

Burkholderia dinitrotoluene (DNT) dioxygenase in this study (from recombinant Esherichia coli strain PFJS39) is probably a multicomponent enzyme system that oxidizes 2,4-dinitrotoluene (DNT) to 4-methyl-5-nitrocatechol (MNC). DNT dioxygenase was purified by a four-step procedure that utilized consecutive gel filtration chromatography and a nondenaturing gel system. The purified enzyme oxidized DNT only in the presence of NADH and its yield increased by lipase pretreatment of crude cytosol. An estimated molecular weight of 100,000 was obtained by gel filtration. Polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) revealed the presence of three subunits for the samples from consecutive gel filtration chromatography and nondenaturing PAGE. Their molecular weights were 52,000–71,000, 23,000–25,500, and 12,000–16,500. These results suggest that DNT dioxygenase exists as a heterotrimer. The K _M of DNT dioxygenase for O₂ is 50 μ M, consistent with inhibition results of DNT dioxygenase by Vitreoscilla hemoglobin (its K _M for O₂ is 7 μ M). The K _M for DNT is 180 μ M. The purified enzyme is relatively stable below 40°C, retains activity over a broad pH range, and is stimulated by several cofactors in addition to NADH.

KEYWORDS:

• dioxygenase
• 2,4-DNT
• purification

ACKNOWLEDGMENTS

The author would like to thank Professors Dale A. Webster and Ben Stark from the Illinois Institute of Technology, Chicago, IL, for their help in discussing this work.