title: Protein engineering of the 4-methyl-5-nitrocatechol monooxygenase from Burkholderia sp. strain DNT for enhanced degradation of nitroaromatics.
abstract: 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 microM 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 microM, 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.
authors.affiliation: Artie McFerrin Department of Chemical Engineering, 220 Jack E. Brown Building, Texas A&M University, College Station, TX 77843-3122, USA.
authors.forename: Thammajun
authors.initials: T
authors.lastname: Leungsakul
authors.forename: Glenn R
authors.initials: GR
authors.lastname: Johnson
authors.forename: Thomas K
authors.initials: TK
authors.lastname: Wood
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D001673:Biodegradation, Environmental; D019117:Burkholderia / Q000201:enzymology*; D017931:DNA Primers; D014644:Genetic Variation; D006899:Mixed Function Oxygenases / Q000235:genetics*; D008969:Molecular Sequence Data; D009573:Nitrites / Q000378:metabolism*; D009596:Nitrophenols / Q000493:pharmacokinetics*; D015202:Protein Engineering / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D009573:Nitrites; D009596:Nitrophenols; C512858:4-methyl-5-nitrocatechol monooxygenase, Burkholderia; D006899:Mixed Function Oxygenases