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

SUBMITTER: Leungsakul T 

PROVIDER: S-EPMC1489588 | biostudies-literature | 2006 Jun

REPOSITORIES: biostudies-literature