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Applied and Environmental Microbiology, June 2006, p. 3933-3939, Vol. 72, No. 6
0099-2240/06/$08.00+0     doi:10.1128/AEM.02966-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Protein Engineering of the 4-Methyl-5-Nitrocatechol Monooxygenase from Burkholderia sp. Strain DNT for Enhanced Degradation of Nitroaromatics

Thammajun Leungsakul,1,{dagger} Glenn R. Johnson,2 and Thomas K. Wood1*

Artie McFerrin Department of Chemical Engineering and the Departments of Biology and Civil/Environmental Engineering, 220 Jack E. Brown Building, Texas A&M University, College Station, Texas 77843-3122,1 Air Force Research Laboratory, U.S. Air Force, Tyndall Air Force Base, Florida 324032

Received 15 December 2005/ Accepted 22 March 2006

4-Methyl-5-nitrocatechol (4M5NC) monooxygenase (DntB) from Burkholderia sp. strain DNT catalyzes the second step of 2,4-dinitrotoluene degradation by converting 4M5NC to 2-hydroxy-5-methylquinone with the concomitant removal of the nitro group. DntB is a flavoprotein that has a very narrow substrate range. Here, error-prone PCR was used to create variant DntB M22L/L380I, which accepts the two new substrates 4-nitrophenol (4NP) and 3-methyl-4-nitrophenol (3M4NP). At 300 µM of 4NP, the initial rate of the variant expressing M22L/L380I enzyme (39 ± 6 nmol/min/mg protein) was 10-fold higher than that of the wild-type enzyme (4 ± 2 nmol/min/mg protein). The values of kcat/Km of the purified wild-type DntB enzyme and purified variant M22L/L380I were 40 and 450 (s–1 M–1), respectively, which corroborates that the variant M22L/L380I enzyme has 11-fold-higher efficiency than the wild-type enzyme for 4NP degradation. In addition, the variant M22L/L380I enzyme has fourfold-higher activity toward 3M4NP; at 300 µM, the initial nitrite release rate of M22L/L380I enzyme was 17 ± 4 nmol/min/mg protein, while that of the wild-type enzyme was 4.4 ± 0.7 nmol/min/mg protein. Saturation mutagenesis was also used to further investigate the role of the individual amino acid residues at positions M22, L380, and M22/L380 simultaneously. Mutagenesis at the individual positions M22L and L380I did not show appreciable enhancement in 4NP activity, which suggested that these two sites should be mutated together; simultaneous saturation mutagenesis led to the identification of the variant M22S/L380V, with 20% enhanced degradation of 4NP compared to the variant M22L/L380I. This is the first report of protein engineering for nitrite removal by a flavoprotein.

* Corresponding author. Mailing address: Artie McFerrin Department of Chemical Engineering and the Departments of Biology and Civil/Environmental Engineering, 220 Jack E. Brown Building, Texas A&M University, College Station, TX 77843-3122. Phone: (979) 862-1588. Fax: (979) 865-6446. E-mail: Thomas.Wood{at}chemail.tamu.edu.

{dagger} Present address: Sirindhorn International Institute of Technology, School of Bio-Chemical Engineering and Technology, Thammasat University, P.O. Box 22, Thammasat Rangsit Post Office, Pathumthani 12121, Thailand.

Applied and Environmental Microbiology, June 2006, p. 3933-3939, Vol. 72, No. 6
0099-2240/06/$08.00+0     doi:10.1128/AEM.02966-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Sanchez-Torres, V., Maeda, T., Wood, T. K. (2009). Protein Engineering of the Transcriptional Activator FhlA To Enhance Hydrogen Production in Escherichia coli. Appl. Environ. Microbiol. 75: 5639-5646 [Abstract] [Full Text]  


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