Enzymes of a New Modified ortho-Pathway Utilizing 2-Chlorophenol in Rhodococcus opacus 1CP

O. V. Moiseeva¹, O. V. Belova¹, I. P. Solyanikova¹, M. Schlömann², and L. A. Golovleva^1*

¹Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region, 142290 Russia; fax: (095) 923-3602; E-mail: Golovleva@ibpm.serpukhov.su

²TU-Bergakademie Freiberg, IOZ, Freiberg, 09596, Germany

^* To whom correspondence should be addressed.

Received October 20, 2000; Revision received February 5, 2001

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Chlorocatechol 1,2-dioxygenase (CC 1,2-DO), chloromuconate cycloisomerase (CMCI), chloromuconolactone isomerase (CMLI), and dienolactone hydrolase (DELH), the key enzymes of a new modified ortho-pathway in Rhodococcus opacus 1CP cells utilizing 2-chlorophenol via a 3-chlorocatechol branch of a modified ortho-pathway, were isolated and characterized. CC 1,2-DO showed the maximum activity with 3-chlorocatechol; its activity with catechol and 4-chlorocatechol was 93 and 50%, respectively. The enzyme of the studied pathway had physicochemical properties intermediate between the pyrocatechase of ordinary and chlorocatechase of modified pathways described earlier for this strain. In contrast to the enzymes investigated earlier, CMCI of the new pathway exhibited high substrate specificity. The enzyme had K_m for 2-chloromuconate of 142.86 µM, V_max = 71.43 U/mg, pH optimum around 6.0, and temperature optimum at 65°C. CMCI converted 2-chloromuconate into 5-chloromuconolactone. CMLI converted 5-chloromuconolactone into cis-dienolactone used as a substrate by DELH; this enzyme did not convert trans-dienolactone. DELH had K_m for cis-dienolactone of 200 µM, V_max = 167 U/mg, pH optimum of 8.6, and temperature optimum of 40°C. These results confirm the existence of a new modified ortho-pathway for utilization of 2-chlorophenol by R. opacus 1CP.

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KEY WORDS: modified ortho-pathway, chlorocatechol 1,2-dioxygenase, chloromuconate cycloisomerase, chloromuconolactone isomerase, dienolactone hydrolase, 2-chlorophenol, Rhodococcus opacus 1CP

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