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.
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.
Kaulmann
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U
Institut für Mikrobiologie, Universität Stuttgart, D-70569 Stuttgart, Germany.
Kaschabek
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SR
Schlömann
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M
D002384:Catalysis; D002712:Chlorides / Q000378:metabolism*; D014644:Genetic Variation; D019753:Intramolecular Lyases / Q000235:genetics / Q000378:metabolism*; D007783:Lactones / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D016958:Pseudomonas putida; D013011:Sorbic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*
C432473:2,4-dichloro-muconate; C432474:2-chloroprotoanemonin; C432472:3-chloro-muconate; D002712:Chlorides; D007783:Lactones; D019753:Intramolecular Lyases; C021811:muconate cycloisomerase; C055773:chloromuconate cycloisomerase; D013011:Sorbic Acid