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

doi:10.1128/JB.183.15.4551-4561.2001

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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^, 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-muconateto cis-dienelactone (cis-4-carboxymethylenebut-2-en-4-olide),while usual muconate cycloisomerases transform the same substrateto the bacteriotoxic protoanemonin. Formation of protoanemoninrequires that the cycloisomerization of 3-chloro-cis,cis-muconateto 4-chloromuconolactone is completed by protonation of the exocycliccarbon of the presumed enol/enolate intermediate before chlorideelimination and decarboxylation take place to yield the finalproduct. The formation of cis-dienelactone, in contrast, couldoccur 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 chlorideelimination, the proton-donating Lys169 of Pseudomonas putidamuconate cycloisomerase was changed to alanine. As expected, substratesrequiring protonation, such as cis,cis-muconate as well as 2-and 3-methyl-, 3-fluoro-, and 2-chloro-cis,cis-muconate, werenot 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-dienelactonewere formed as products, whereas the wild-type enzyme forms protoanemoninand the not previously isolated 2-chloroprotoanemonin, respectively.Thus, the chloromuconate cycloisomerases may avoid (chloro-)protoanemoninformation by increasing the rate of chloride abstraction fromthe enol/enolate intermediate compared to that of proton additiontoit.

^* Corresponding author. Present address: TU Bergakademie Freiberg, Interdisziplinäres Ökologisches Zentrum, Leipziger Str.29, D-09599 Freiberg, Germany. Phone: 49 3731 39 3739. Fax: 493731 39 3012. E-mail: michael.schloemann{at}ioez.tu-freiberg.de.

Dedicated to Hans-Joachim Knackmuss on the occasion of his 65thbirthday.

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

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

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