Kinetics of hydrolysis and mutational analysis ofN,N-diethyl-m-toluamide hydrolase fromPseudomonas putidaDTB

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Abstract

The initial step in the biodegradation pathway of N, N-diethyl-m-toluamide (DEET) in Pseudomonas putida strain DTB is catalyzed by DEET hydrolase (DthA), which hydrolyzes the amide bond to yield 3-methylbenzoic acid and diethylamine. In order to extend our understanding of DthA, the enzyme was purified and characterized. The enzyme is most active at pH 7.9, and is probably a tetramer in its native state. The kinetic parameters of the wild-type enzyme are Km = 10.2 ± 0.8 μm, kcat = 5.53 ± 0.09 s−1, and kcat/Km = (5.4 ± 0.4) × 105 m−1·s−1. Mild substrate inhibition was observed with DEET concentrations over 500 μm. A homology model of DthA was used to guide mutational analysis of the active site, confirming that the catalytic triad is formed by Ser166, Ap292, and His320. The oxyanion hole is formed by the side chain OH of Tyr84 and the backbone amide of Trp167, with the Tyr84 OH being essential for enzyme activity. The DthA model also revealed a hydrophobic substrate-binding pocket comprosed of Trp167, Met170, and Trp214. W167A and M170A mutations decreased enzymatic activity and exacerbated substrate inhibition, whereas Trp214, which probably plays a role in substrate recognition, was essential for enzymatic activity. The pH rate profile of DthA was fitted to two ionizable groups (pKa1 = 6.1 and pKa2 = 9.9) that probably correspond to Nε of His320 and the OH of Tyr84, respectively. In addition to catalyzing the hydrolysis of DEET, DthA hydrolyzed a variety of esters and amides.

Database

Model data are available in the PMDB database under the accession number PM0077728.

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