Characterization and evolution of anthranilate 1,2-dioxygenase from Acinetobacter sp. strain ADP1

Area of Science:

• Biochemistry
• Microbiology
• Enzymology

Background:

• Anthranilate 1,2-dioxygenase is a key enzyme in microbial degradation pathways.
• Understanding its structure and function is crucial for bioremediation strategies.

Purpose of the Study:

• To express, purify, and characterize the two-component anthranilate 1,2-dioxygenase from Acinetobacter sp. strain ADP1.
• To investigate the enzyme's catalytic mechanism, cofactor content, and substrate specificity.
• To analyze a temperature-sensitive variant and compare its activity to the wild-type enzyme.

Main Methods:

• Heterologous expression in Escherichia coli and protein purification.
• Biochemical assays to measure enzyme activity, cofactor content, and substrate conversion.
• Characterization of a specific enzyme variant (M43K) under varying temperatures.
• Phylogenetic analysis of enzyme subunits.

Main Results:

• The enzyme was purified, revealing a heterohexameric oxygenase component with Rieske [2Fe-2S] and nonheme iron centers, and a reductase component with FAD and ferredoxin [2Fe-2S] centers.
• Anthranilate conversion to catechol and benzoate to benzoate 1,2-diol were tightly coupled to NADH oxidation and O(2) consumption.
• The M43K variant showed no catalytic activity at permissive or nonpermissive temperatures.
• Wild-type enzyme exhibited limited activity on methylated or halogenated benzoates.

Conclusions:

• The purified anthranilate 1,2-dioxygenase functions as a two-component system with specific cofactor requirements.
• The enzyme's activity is tightly coupled to oxygen and NADH consumption, indicating efficient catalysis.
• The M43K mutation abolishes enzyme activity, suggesting its critical role in enzyme function.
• Enzyme evolution indicates common ancestry for dioxygenases acting on diverse substrates.