Novel Function of CtXyn5A from Acetivibrio thermocellus: Dual Arabinoxylanase and Feruloyl Esterase Activity in the Same Active Site

Enzymes' uncharacterised side activities can have significant effects on reaction products and yields. Hence, their identification and characterisation are crucial for the development of successful reaction systems. Here, we report the presence of feruloyl esterase activity in CtXyn5A from Acetivibrio thermocellus, besides its well-known arabinoxylanase activity, for the first time. Activity analysis of enzyme variants mutated in the catalytic nucleophile, Glu279, confirmed removal of all activity for E279A and E279L, and increased esterase activity while removing xylanase activity for E279S, thus allowing the proposal that both reaction types are catalysed in the same active site in two subsequential steps. The ferulic acid substituent is cleaved off first, followed by hydrolysis of the xylan backbone. The esterase activity on complex carbohydrates was found to be higher than that of a designated ferulic acid esterase (E-FAERU). Therefore, we conclude that the enzyme exhibits a dual function rather than an esterase side activity.

Automated summary

In this study, we discovered that the enzyme CtXyn5A from Acetivibrio thermocellus exhibits both arabinoxylanase and feruloyl esterase activities. Mutation analysis confirmed that the removal of catalytic nucleophile Glu279 abolishes all activity, and the E279S mutation increases esterase activity while removing xylanase activity. This suggests that both reactions are catalyzed in the same active site in two sequential steps. Furthermore, the esterase activity of CtXyn5A on complex carbohydrates is higher than that of a designated ferulic acid esterase, indicating that this enzyme has a dual function rather than a side activity.