Mechanism of Chloride Elimination from 3-Chloro- and 2,4-Dichloro-cis,cis-Muconate: New Insight Obtained from Analysis of Muconate Cycloisomerase Variant CatB-K169A

ABSTRACT Chloromuconate cycloisomerases of bacteria utilizing chloroaromatic compounds are known to convert 3-chloro-cis,cis-muconate tocis-dienelactone (cis-4-carboxymethylenebut-2-en-4-olide), while usual muconate cycloisomerases transform the same substrate to the bacteriotoxic protoanemonin. Formation of protoanemonin requires that the cycloisomerization of 3-chloro-cis,cis-muconate to 4-chloromuconolactone is completed by protonation of the exocyclic carbon of the presumed enol/enolate intermediate before chloride elimination and decarboxylation take place to yield the final product. The formation ofcis-dienelactone, in contrast, could occur either by dehydrohalogenation of 4-chloromuconolactone or, more directly, by chloride elimination from the enol/enolate intermediate. To reach a better understanding of the mechanisms of chloride elimination, the proton-donating Lys169 of Pseudomonas putida muconate cycloisomerase was changed to alanine. As expected, substrates requiring protonation, such ascis,cis-muconate as well as 2- and 3-methyl-, 3-fluoro-, and 2-chloro-cis,cis-muconate, were not converted at a significant rate by the K169A variant. However, the variant was still active with 3-chloro- and 2,4-dichloro-cis,cis-muconate. Interestingly,cis-dienelactone and 2-chloro-cis-dienelactone were formed as products, whereas the wild-type enzyme forms protoanemonin and the not previously isolated 2-chloroprotoanemonin, respectively. Thus, the chloromuconate cycloisomerases may avoid (chloro-)protoanemonin formation by increasing the rate of chloride abstraction from the enol/enolate intermediate compared to that of proton addition to it.

[1]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[2]  M. Schlömann,et al.  Substrate specificities of the chloromuconate cycloisomerases from Pseudomonas sp. B13, Ralstonia eutropha JMP134 and Pseudomonas sp. P51 , 1999, Applied Microbiology and Biotechnology.

[3]  A. Goldman,et al.  Substrate Specificity of and Product Formation by Muconate Cycloisomerases: an Analysis of Wild-Type Enzymes and Engineered Variants , 1998, Applied and Environmental Microbiology.

[4]  D. Eulberg,et al.  Evolutionary Relationship between Chlorocatechol Catabolic Enzymes from Rhodococcus opacus 1CP and Their Counterparts in Proteobacteria: Sequence Divergence and Functional Convergence , 1998, Journal of bacteriology.

[5]  A. Goldman,et al.  The refined X-ray structure of muconate lactonizing enzyme from Pseudomonas putida PRS2000 at 1.85 A resolution. , 1995, Journal of molecular biology.

[6]  K. Timmis,et al.  From Xenobiotic to Antibiotic, Formation of Protoanemonin from 4-Chlorocatechol by Enzymes of the 3-Oxoadipate Pathway (*) , 1995, The Journal of Biological Chemistry.

[7]  M. Schlömann,et al.  Conversion of 2-chloro-cis,cis-muconate and its metabolites 2-chloro- and 5-chloromuconolactone by chloromuconate cycloisomerases of pJP4 and pAC27 , 1995, Journal of bacteriology.

[8]  P. March,et al.  Easy Access to 5-Alkyl-4-bromo-2(5H)-furanones: Synthesis of a Fimbrolide, an Acetoxyfimbrolide, and Bromobeckerelide , 1995 .

[9]  L. N. Ornston,et al.  Copyright � 1995, American Society for Microbiology Discontinuities in the Evolution of Pseudomonas putida cat Genes† , 1994 .

[10]  M. Schlömann,et al.  Inability of muconate cycloisomerases to cause dehalogenation during conversion of 2-chloro-cis,cis-muconate , 1994, Journal of bacteriology.

[11]  S. Michael,et al.  Mutagenesis by incorporation of a phosphorylated oligo during PCR amplification. , 1994, BioTechniques.

[12]  K. H. Kalk,et al.  Crystallographic and fluorescence studies of the interaction of haloalkane dehalogenase with halide ions. Studies with halide compounds reveal a halide binding site in the active site. , 1993, Biochemistry.

[13]  P. G. Gassman,et al.  Understanding enzyme-catalyzed proton abstraction from carbon acids: Details of stepwise mechanisms for β-elimination reactions , 1992 .

[14]  D. Pieper,et al.  Metabolization of 3,5-dichlorocatechol by Alcaligenes eutrophus JMP 134 , 1991, Archives of Microbiology.

[15]  Meer,et al.  Sequence analysis of the Pseudomonas sp. strain P51 tcb gene cluster, which encodes metabolism of chlorinated catechols: evidence for specialization of catechol 1,2-dioxygenases for chlorinated substrates , 1991, Journal of bacteriology.

[16]  M. Schlömann,et al.  Enzymatic formation, stability, and spontaneous reactions of 4-fluoromuconolactone, a metabolite of the bacterial degradation of 4-fluorobenzoate , 1990, Journal of bacteriology.

[17]  E. Perkins,et al.  Organization and sequence analysis of the 2,4-dichlorophenol hydroxylase and dichlorocatechol oxidative operons of plasmid pJP4 , 1990, Journal of bacteriology.

[18]  D. Pieper,et al.  Purification and characterization of dichloromuconate cycloisomerase from Alcaligenes eutrophus JMP 134. , 1990, The Biochemical journal.

[19]  Matthew Guille,et al.  A Practical Guide to Molecular Cloning (2nd ed) : by Bernard Perbal Wiley Interscience; New York, 1988 811 pages. £32.50 , 1989 .

[20]  C. A. Fewson.  Biodegradation of xenobiotic and other persistent compounds: the causes of recalcitrance , 1988 .

[21]  J. Kozarich,et al.  Absolute stereochemical course of the 3-carboxymuconate cycloisomerases from Pseudomonas putida and Acinetobacter calcoaceticus: analysis and implications , 1987 .

[22]  B. Frantz,et al.  Organization and nucleotide sequence determination of a gene cluster involved in 3-chlorocatechol degradation. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[23]  W. Reineke,et al.  Microbial metabolism of haloaromatics: isolation and properties of a chlorobenzene-degrading bacterium , 1984, Applied and environmental microbiology.

[24]  R. Kallen,et al.  Enzymes of the beta-ketoadipate pathway in Pseudomonas putida: primary and secondary kinetic and equilibrium deuterium isotope effects upon the interconversion of (+)-muconolactone to cis,cis-muconate catalyzed by cis,cis-muconate cycloisomerase. , 1983, Biochemistry.

[25]  H. Knackmuss,et al.  Chemical structure and biodegradability of halogenated aromatic compounds. Conversion of chlorinated muconic acids into maleoylacetic acid. , 1980, The Biochemical journal.

[26]  H. Knackmuss,et al.  Chemical structure and biodegradability of halogenated aromatic compounds. Substituent effects on 1,2-dioxygenation of catechol. , 1978, The Biochemical journal.

[27]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[28]  H. N. Fernley,et al.  Bacterial metabolism of 4-chlorophenoxyacetate. , 1971, The Biochemical journal.

[29]  H. N. Fernley,et al.  Bacterial metabolism of 2,4-dichlorophenoxyacetate. , 1971, The Biochemical journal.

[30]  J. Duxbury,et al.  2,4-D metabolism: pathway of degradation of chlorocatechols by Arthrobacter sp. , 1969, Journal of agricultural and food chemistry.

[31]  L. N. Ornston,et al.  The Conversion of Catechol and Protocatechuate to β-Ketoadipate by Pseudomonas putida I. BIOCHEMISTRY , 1966 .

[32]  Ornston Ln.  The conversion of catechol and protocatechuate to beta-ketoadipate by Pseudomonas putida. II. Enzymes of the protocatechuate pathway. , 1966 .

[33]  R. Stanier,et al.  The mechanism of formation of beta-ketoadipic acid by bacteria. , 1954, The Journal of biological chemistry.

[34]  M. Holden,et al.  THE ANTIBIOTIC ACTIVITY OF EXTRACTS OF RANUNCULACEAE. , 1945, Science.

[35]  A. Goldman,et al.  Structural basis for the activity of two muconate cycloisomerase variants toward substituted muconates , 1999, Proteins.

[36]  M. Alting-Mees,et al.  pBluescriptII: multifunctional cloning and mapping vectors. , 1992, Methods in enzymology.

[37]  F. Studier,et al.  Use of T7 RNA polymerase to direct expression of cloned genes. , 1990, Methods in enzymology.

[38]  H. Nojima,et al.  High efficiency transformation of Escherichia coli with plasmids. , 1990, Gene.

[39]  B. Perbal.  A practical guide to molecular cloning , 1988 .

[40]  T. Leisinger,et al.  Microbial degradation of xenobiotics and recalcitrant compounds , 1981 .

[41]  E. Galli,et al.  (+)-γ-Carboxymethyl-γ-methyl-Δα-butenolide. A 1,2 ring-fission product of 4-methylcatechol by Pseudomonas desmolyticum , 1971 .

[42]  L. N. Ornston,et al.  The conversion of catechol and protocatechuate to beta-ketoadipate by Pseudomonas putida. 3. Enzymes of the catechol pathway. , 1966, The Journal of biological chemistry.