Title

Protein engineering for the degradation of nitroaromatic compounds

Date of Completion

January 2006

Keywords

Chemistry, Biochemistry|Engineering, Chemical

Degree

Ph.D.

Abstract

2,4-Dinitrotoluene (2,4-DNT) dioxygenases from Burkholderia sp. strain DNT (DNT DDO) and Burkholderia cepacia R34 (R34 DDO) catalyze the first step of the aerobic degradation of 2,4-DNT. They convert 2,4-DNT to 4-methyl-5 nitrocatechol.(4M5NC) with the release of nitrite by adding both atoms of oxygen. This allows these bacteria to use 2,4-DNT as sole carbon and nitrogen sources. 4M5NC monooxygenase (DntB) catalyzes the second step in 2,4-DNT degradation. It converts 4M5NC to 2-hydroxy-5-methylquinone with the release of nitrite and uses flavin adenine dinucleotide as a cofactor. DntB has a very narrow substrate range since there is only one known substrate, 4-nitrocatechol, in addition to the natural substrate 4M5NC. ^ Tri-, di-, and mononitrotoluenes are toxic compounds, but they have extremely important roles in the chemical industry. Trinitrotoluene is the most-widely-used nitroaromatic in military operations. It is a very stable and recalcitrant compound. Dinitrotoluenes are also contaminants in soil and ground water. They are intermediates in TNT production. In addition, mononitrotoluenes such as o- and p-nitrotoluene are on the Environmental Protection Agency for high production chemicals which should be limited in terms of human exposure. o-Nitrotoluene is a carcinogen for mice. ^ Directed evolution, saturation mutagenesis, and site-directed mutagenesis were used in this work to engineer DNT DDO, R34 DDO, and DNT DntB to enhance the catalytic activity of the enzymes for nitroaromatic compound degradation and to expand their substrate range. Variants 1204L and 1204Y of DntAc DNT DDO were created to degrade 2,3-DNT and 2,5-DNT for the first time since there was no detectable activity with the wild-type enzyme; this is the first protein engineering for nitroaromatic degradation. ^ Variant S349C/T350F of DntAc DNT DDO was also found that has enhanced oxidization of substituted phenols and catechols (including indole) to produce useful compounds such as methoxyhydroquinone, nitrohydroquinone, and indigoid compounds. In addition, a variant M22L/L380I of DntB was generated using error prone PCR. This variant has an enhanced substrate range as it was engineered to accept the new substrates 4-nitrophenol (4NP) and 3-methyl-4-nitrophenol (3M4NP) which are major toxic contaminants from industry and agriculture. Both the wild-type and variant were purified to show that there is 11-fold greater activity of the variant with 4NP. This is the first successful directed evolution of an enzyme for nitrite removal by a flavoprotein. ^ To facilitate nitroaromatic biodegradation, Burkholderia cepacia R34 and Burkholderia sp. strain DNT were identified that have chemotaxis toward TNT, 2,3-DNT, 2,4-DNT, 2,5-DNT, 2-nitrotoluene (NT), 4NT, and 4M5NC but not toward 2,6-DNT. (Abstract shortened by UMI.)^