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J. Biol. Chem., Vol. 278, Issue 15, 12779-12785, April 11, 2003

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Purification and Characterization of PrbA, a New Esterase from Enterobacter cloacae Hydrolyzing the Esters of 4-Hydroxybenzoic Acid (Parabens)^*

Nelly Valkova, François Lépine, Louisette Labrie, Maryse Dupont, and Réjean Beaudet

From the Institut Armand-Frappier, Institut National de la Recherche Scientifique, Université du Québec, Laval, Québec H7V 1B7, Canada

The esterase PrbA from Enterobacter cloacae strain EM has previously been shown to confer additional resistance to the esters of 4-hydroxybenzoic acid (parabens) to two species of Enterobacter. The PrbA protein has been purified from E. cloacae strain EM using a three-step protocol resulting in a 60-fold increase in specific activity. The molecular mass of the mature enzyme was determined to be 54,619 ± 1 Da by mass spectrometry. It is highly active against a series of parabens with alkyl groups ranging from methyl to butyl, with K_m and V_max values ranging from 0.45 to 0.88 mM and 0.031 to 0.15 mM/min, respectively. The K_m and V_max values for p-nitrophenyl acetate were 3.7 mM and 0.051 mM/min. PrbA hydrolyzed a variety of structurally analogous compounds, with activities larger than 20% relative to propyl paraben for methyl 3-hydroxybenzoate, methyl 4-aminobenzoate, or methyl vanillate. The enzyme showed optimum activity at 31 °C and at pH 7.0. PrbA was able to transesterify parabens with alcohols of increasing chain length from methanol to n-butanol, achieving 64% transesterification of 0.5 mM propyl paraben with 5% methanol within 2 h. PrbA was inhibited by 1-chloro-3-tosylamido-4-phenyl-2-butanone and 1-chloro-3-tosylamido-7- amino-2-heptanone (TLCK), with K_i values of 0.29 and 0.20 mM, respectively, and was irreversibly inhibited by Diisopropyl fluorophosphate (DFP) or diethyl pyrocarbonate. The stoichiometry of addition of DFP to the enzyme was 1:1 and only 1 TLCK molecule was found in TLCK-modified enzyme, as measured by mass spectrometry. Analysis of the tryptic digest of the DFP-modified PrbA demonstrated that the addition of a DFP molecule occurred at Ser-189, indicating the location of the active serine.

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