Dienelactone hydrolase from Pseudomonas sp. strain B13.
Dienelactone hydrolase (EC 3.1.1.45) catalyzes the conversion of cis- or trans-4-carboxymethylenebut-2-en-4-olide (dienelactone) to maleylacetate. An approximately 24-fold purification from extracts of 3-chlorobenzoate-grown Pseudomonas sp. strain B13 yielded a homogeneous preparation of the enzyme. The purified enzyme crystallized readily and proved to be a monomer with a molecular weight of about 30,000. Each dienelactone hydrolase molecule contains two cysteinyl side chains. One of these was readily titrated by stoichiometric amounts of p-chloromercuribenzoate, resulting in inactivation of the enzyme; the inactivation could be reversed by the addition of dithiothreitol. The other cysteinyl side chain appeared to be protected in the native protein against chemical reaction with p-chloromercuribenzoate. The properties of sulfhydryl side chains in dienelactone hydrolase resembled those that have been characterized for bacterial 4-carboxymethylbut-3-en-4-olide (enol-lactone) hydrolases (EC 3.1.1.24), which also are monomers with molecular weights of about 30,000. The amino acid composition of the dienelactone hydrolase resembled the amino acid composition of enol-lactone hydrolase from Pseudomonas putida, and alignment of the NH2-terminal amino acid sequence of the dienelactone hydrolase with the corresponding sequence of an Acinetobacter calcoaceticus enol-lactone hydrolase revealed sequence identity at 8 of the 28 positions. These observations foster the hypothesis that the lactone hydrolases share a common ancestor. The lactone hydrolases differed in one significant property: the kcat of dienelactone hydrolase was 1,800 min-1, an order of magnitude below the kcat observed with enol-lactone hydrolases. The relatively low catalytic activity of dienelactone hydrolase may demand its production at the high levels observed for induced cultures of Pseudomonas sp. strain B13.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002265:Carboxylic Ester Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D002729:Chloromercuribenzoates / Q000494:pharmacology; D007700:Kinetics; D011549:Pseudomonas / Q000201:enzymology*; D020245:p-Chloromercuribenzoic Acid
D000596:Amino Acids; D002729:Chloromercuribenzoates; D020245:p-Chloromercuribenzoic Acid; D002265:Carboxylic Ester Hydrolases; C046130:carboxymethylenebutenolidase