Engineering the catechol 1,2-dioxygenase from Arthrobacter sp. with improved activity toward 4-alkyl substituted catechols

Catechol 1,2-dioxygenase (CAT) is a key enzyme in the cis, cis-muconic acid biosynthesis pathways. However, CAT demonstrates limited catalytic ability to the substrate 4-alkyl substituted catechols, and its application in biocatalysis remains underexplored. Here, a CAT from Arthrobacter sp. CGMCC 3584 (ArCAT) was overexpressed in Escherichia coli for the first time. Subsequently, the structure-guided semi-rational engineering of ArCAT was employed to generate small and precise mutation libraries, and the mutant I206V exhibited the best catalytic ability. Molecular docking analysis revealed that the mutant I206V provided additional hydrophobic interactions and hydrogen bonds with the substrates in the catalytic activity pocket compared with the wild type (WT) for both 4-ethylcatechol and 4-n-propylcatechol. Furthermore, binding free energy calculations showed that the mutant I206V had a better binding affinity to 4-ethylcatechol or to 4-n-propylcatechol compared to the WT, in good agreement with the results of kinetic parameters. Finally, in small-scale biotransformation based on crude enzymes, the mutant I206V enhanced the overall conversion rates of 4-ethylcatechol and 4-n-propylcatechol by 15.28 % and 25.55 %, respectively, compared to the WT. This study presented a smart strategy for improving ArCAT activity, and revealed the potential application of ArCAT in biological production of muconic acid analogs from branched catechols.