Substrate specificities of the chloromuconate cycloisomerases from Pseudomonas sp. B13, Ralstonia eutropha JMP134 and Pseudomonas sp. P51.
The chloromuconate cycloisomerase of Pseudomonas sp. B13 was purified from 3-chlorobenzoate-grown wild-type cells while the chloromuconate cycloisomerases of Ralstonia eutropha JMP134 (pJP4) and Pseudomonas sp. P51 (pP51) were purified from Escherichia coli strains expressing the corresponding gene. Kinetic studies were performed with various chloro-, fluoro-, and methylsubstituted cis,cis-muconates. 2,4-Dichloro-cis,cis-muconate proved to be the best substrate for all three chloromuconate cycloisomerases. Of the three enzymes, TfdD of Ralstonia eutropha JMP134 (pJP4) was most specific, since its specificity constant for 2,4-dichloro-cis,cis-muconate was the highest, while the constants for cis,cis-muconate, 2-chloro- and 2,5-dichloro-cis,cis-muconate were especially poor. The sequence of ClcB of the 3-chloro-benzoate-utilizing strain Pseudomonas sp. B13 was determined and turned out to be identical to that of the corresponding enzyme of pAC27 (though slightly different from the published sequences). Corresponding to 2-chloro-cis,cis-muconate being a major metabolite of 3-chlorobenzoate degradation, the kcat/K(m) with 2-chloro-cis,cis-muconate was relatively high, while that with the still preferred substrate 2,4-dichloro-cis,cis-muconate was relatively low. This enzyme was thus the least specific and the least active among the three compared enzymes. TcbD of Pseudomonas sp. P51 (pP51) took an intermediate position with respect to both the degree of specificity and the activity with the preferred substrate.
Vollmer
M D
MD
Institut für Mikrobiologie, Universität Stuttgart, Germany.
Schell
U
U
Seibert
V
V
Lakner
S
S
Schlömann
M
M
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D018533:Biomass; D003001:Cloning, Molecular; D020118:Cupriavidus necator / Q000201:enzymology*; D019753:Intramolecular Lyases / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011549:Pseudomonas / Q000201:enzymology*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D019753:Intramolecular Lyases; C055773:chloromuconate cycloisomerase