Purification and characterization of PrbA, a new esterase from Enterobacter cloacae hydrolyzing the esters of 4-hydroxybenzoic acid (parabens).
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 degrees 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.
Valkova
Nelly
N
Institut Armand-Frappier, Institut National de la Recherche Scientifique, Université du Québec, Laval, Québec H7V 1B7, Canada.
Lépine
François
F
Labrie
Louisette
L
Dupont
Maryse
M
Beaudet
Réjean
R
D000595:Amino Acid Sequence; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D016972:Enterobacter cloacae / Q000201:enzymology*; D004795:Enzyme Stability; D004950:Esterases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010226:Parabens / Q000493:pharmacokinetics; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity; D013816:Thermodynamics; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology
D010226:Parabens; D010446:Peptide Fragments; D014107:Tosyllysine Chloromethyl Ketone; D004950:Esterases; D002265:Carboxylic Ester Hydrolases; C475057:PrbA protein, Enterobacter cloacae