Functional Characterization of a Cold-Active Catechol 2,3- Dioxygenase from an Antarctic Pseudomonas Strain

Abstract

The eurypsychrophilic Antarctic bacterium Pseudomonas strain MC1 KOPRI 26573 metabolizes naphthalene through salicylate and catechol, which is further cleaved by a catechol 2,3- dioxygenase. A putative gene encoding this enzyme (nahH) was amplified by PCR, cloned, and expressed in Escherichia coli. Assessment of the enzyme activity expressed in E. coli demonstrated that the nahH gene encodes the bona fide catechol 2,3-dioxygenase (NahH) for metabolism of naphthalene and salicylate. The NahH produced in E. coli BL21(DE3) has maximal activity against catechol, significant activity against 4-methylcatechol (70% of that against catechol), and modest activity against 3-methylcatechol (45% of that against catechol). It is noteworthy that the ratios of specific activity measured at 5°C to that at 30°C were 0.7. However, preincubation of the assay mixture without substrate at different temperatures has a dramatically different effect on enzyme activity. Namely, the higher the incubation temperature, the more rapid decays of enzyme activity were observed. 30-min and one-hour preincubations at 30°C resulted in 60 and 98% loss of enzyme activity, respectively, while preincubation at 0°C did not show any negative effect on the initial enzyme activity. These data indicate that the thermounstability of the NahH enzyme is one reason for our previous observation that while MC1 grows fine on succinate as amplified by PCR, cloned, and expressed in Escherichia coli. Assessment of the enzyme activity expressed in E. coli demonstrated that the nahH gene encodes the bona fide catechol 2,3-dioxygenase (NahH) for metabolism of naphthalene and salicylate. The NahH produced in E. coli BL21(DE3) has maximal activity against catechol, significant activity against 4-methylcatechol (70% of that against catechol), and modest activity against 3-methylcatechol (45% of that against catechol). It is noteworthy that the ratios of specific activity measured at 5°C to that at 30°C were 0.7.