Catechol 2,3-dioxygenase isoenzymes from Diaphorabacter sp. strain DS2 demonstrate different substrate preferences

Area of Science:

• Biochemistry
• Enzymology
• Environmental Microbiology

Background:

• Catechol 2,3-dioxygenases (C23O) are key enzymes in aromatic compound degradation.
• Diaphorobacter sp. strain DS2 possesses multiple C23O isozymes with potential roles in pollutant metabolism.

Purpose of the Study:

• To clone, overexpress, and characterize two monomeric catechol 2,3-dioxygenase isozymes (C23O64 and C23O68) from Diaphorobacter sp. strain DS2.
• To investigate the enzymatic properties and substrate specificities of these isozymes.

Main Methods:

• Gene cloning and overexpression of C23O64 and C23O68 in E. coli BL21.
• Enzyme activity assays with various substituted catechols.
• Determination of optimal temperature and effects of inhibitors/enhancers.

Main Results:

• Both C23O64 and C23O68 catalyze the m-cleavage of catechol, producing 2-hydroxymuconic semialdehyde (2-HMS).
• C23O64 showed high activity towards 4-methyl catechol and 3-methyl catechol, while C23O68 preferred 4-chloro catechol.
• Maximal activity for both isozymes was observed at 37°C, with differential responses to various chemical agents.

Conclusions:

• The two isozymes share high nucleotide homology but exhibit distinct substrate specificities for substituted catechols.
• This enzymatic diversity enhances the capability of Diaphorobacter sp. to metabolize complex mixtures of aromatic pollutants.