Search tips
Search criteria 


Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. 1987 February; 169(2): 699–703.
PMCID: PMC211836

Dienelactone hydrolase from Pseudomonas sp. strain B13.


Dienelactone hydrolase (EC 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, 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.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1006K), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Andrews P. Estimation of the molecular weights of proteins by Sephadex gel-filtration. Biochem J. 1964 May;91(2):222–233. [PubMed]
  • Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. [PubMed]
  • Chatterjee DK, Chakrabarty AM. Genetic homology between independently isolated chlorobenzoate-degradative plasmids. J Bacteriol. 1983 Jan;153(1):532–534. [PMC free article] [PubMed]
  • Frantz B, Ngai KL, Chatterjee DK, Ornston LN, Chakrabarty AM. Nucleotide sequence and expression of clcD, a plasmid-borne dienelactone hydrolase gene from Pseudomonas sp. strain B13. J Bacteriol. 1987 Feb;169(2):704–709. [PMC free article] [PubMed]
  • Ghosal D, You IS, Chatterjee DK, Chakrabarty AM. Genes specifying degradation of 3-chlorobenzoic acid in plasmids pAC27 and pJP4. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1638–1642. [PubMed]
  • HIRS CH. The oxidation of ribonuclease with performic acid. J Biol Chem. 1956 Apr;219(2):611–621. [PubMed]
  • Hugli TE, Moore S. Determination of the tryptophan content of proteins by ion exchange chromatography of alkaline hydrolysates. J Biol Chem. 1972 May 10;247(9):2828–2834. [PubMed]
  • Liu TY, Chang YH. Hydrolysis of proteins with p-toluenesulfonic acid. Determination of tryptophan. J Biol Chem. 1971 May 10;246(9):2842–2848. [PubMed]
  • McCorkle GM, Yeh WK, Fletcher P, Ornston LN. Repetitions in the NH2-terminal amino acid sequence of beta-ketoadipate enol-lactone hydrolase from Pseudomonas putida. J Biol Chem. 1980 Jul 10;255(13):6335–6341. [PubMed]
  • Meagher RB, McCorkle GM, Ornston MK, Ornston LN. Inducible uptake system for -carboxy-cis, cis-muconate in a permeability mutant of Pseudomonas putida. J Bacteriol. 1972 Aug;111(2):465–473. [PMC free article] [PubMed]
  • Merrill BM, Williams KR, Chase JW, Konigsberg WH. Photochemical cross-linking of the Escherichia coli single-stranded DNA-binding protein to oligodeoxynucleotides. Identification of phenylalanine 60 as the site of cross-linking. J Biol Chem. 1984 Sep 10;259(17):10850–10856. [PubMed]
  • Ollis DL, Ngai KL. Crystallization and preliminary x-ray crystallographic data of dienelactone hydrolase from Pseudomonas sp. B13. J Biol Chem. 1985 Aug 15;260(17):9818–9819. [PubMed]
  • Ornston LN. The conversion of catechol and protocatechuate to beta-ketoadipate by Pseudomonas putida. 3. Enzymes of the catechol pathway. J Biol Chem. 1966 Aug 25;241(16):3795–3799. [PubMed]
  • Patel RN, Mazumdar S, Ornston LN. Beta-ketoadipate enol-lactone hydrolases I and II from Acinetobacter calcoaceticus. J Biol Chem. 1975 Aug 25;250(16):6567–6567. [PubMed]
  • Reineke W, Knackmuss HJ. Construction of haloaromatics utilising bacteria. Nature. 1979 Feb 1;277(5695):385–386. [PubMed]
  • Schmidt E, Knackmuss HJ. Chemical structure and biodegradability of halogenated aromatic compounds. Conversion of chlorinated muconic acids into maleoylacetic acid. Biochem J. 1980 Oct 15;192(1):339–347. [PubMed]
  • Spector T. Refinement of the coomassie blue method of protein quantitation. A simple and linear spectrophotometric assay for less than or equal to 0.5 to 50 microgram of protein. Anal Biochem. 1978 May;86(1):142–146. [PubMed]
  • Weber K, Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed]
  • Yeh WK, Fletcher P, Ornston LN. Evolutionary divergence of co-selected beta-ketoadipate enol-lactone hydrolases in Acinetobacter calcoaceticus. J Biol Chem. 1980 Jul 10;255(13):6342–6346. [PubMed]
  • Yeh WK, Ornston LN. p-Chloromercuribenzoate specifically modifies thiols associated with the active sites of beta-ketoadipate enol-lactone hydrolase and succinyl CoA: beta-ketoadipate CoA transferase. Arch Microbiol. 1984 Jun;138(2):102–105. [PubMed]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)