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Journal of Bacteriology, August 2001, p. 4551-4561, Vol. 183, No. 15
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.15.4551-4561.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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†

Ursula Kaulmann,1,Dagger Stefan R. Kaschabek,2,§ and Michael Schlömann1,*

Institut für Mikrobiologie, Universität Stuttgart, D-70569 Stuttgart,1 and Chemische Mikrobiologie, Bergische Universität---Gesamthochschule Wuppertal, D-42097 Wuppertal,2 Germany

Received 16 January 2001/Accepted 15 May 2001

Chloromuconate cycloisomerases of bacteria utilizing chloroaromatic compounds are known to convert 3-chloro-cis,cis-muconate to cis-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 of cis-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 as cis,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.

* Corresponding author. Present address: TU Bergakademie Freiberg, Interdisziplinäres Ökologisches Zentrum, Leipziger Str. 29, D-09599 Freiberg, Germany. Phone: 49 3731 39 3739. Fax: 49 3731 39 3012. E-mail: michael.schloemann{at}ioez.tu-freiberg.de.

dagger Dedicated to Hans-Joachim Knackmuss on the occasion of his 65th birthday.

Dagger Present address: Hans-Knöll-Institut für Naturstoff-Forschung, D-07745 Jena, Germany.

§ Present address: TU Bergakademie Freiberg, Interdisziplinäres Ökologisches Zentrum, D-09599 Freiberg, Germany.

Journal of Bacteriology, August 2001, p. 4551-4561, Vol. 183, No. 15
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.15.4551-4561.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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