The K428 residue from Thermus thermophilus SG0.5JP17-16 laccase plays the substantial role in substrate binding and oxidation

Abstract

Laccases are multi-copper oxidases (MCOs) that catalyze the oxidation of various organic and inorganic compounds with concomitant reduction of dioxygen to water, and are considered to be green catalysts. Previous studies revealed that in MCOs, the structure of substrate-binding pocket was closely related to the substrate specificity. The amino acids on the pocket-constructing loops were involved in the identification of substrate and affected the catalytic properties of the enzyme. In the Thermus thermophilus SG0.5JP17-16 laccase (lacTT), the Lys428 residue is on pocket-constructing loops, to explore the role of the K428 residue and obtain mutants with enhanced catalytic activity, it was mutated to leucine, glutamic acid, arginine and methionine by site-directed mutagenesis. Structural data revealed that the K428 residue identified the conformation of the substrate by the steric hindrance and interactions with the substrate. Kinetic data indicated that the replacement of K428 by methionine resulted in a mutant with enhanced catalytic activity. The K428M mutant could decolorize the synthetic dyes more efficiently than the wild type enzyme. Altogether, this study provided a strategy to find the mutant with the enhanced catalytic activity.

Communicated by Ramaswamy H. Sarma

Keywords:

• Laccase
• kinetic assay
• site-directed mutagenesis
• substrate conformation
• enhanced activity

Compliance with ethical standards

This article does not contain any studies with human participants or animals by any of the authors.

Disclosure statement

The authors declare that they have no competing interests.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China (No. 41673074) to X. Yang.