Applied Microbiology and Biotechnology

, Volume 102, Issue 9, pp 4049–4061 | Cite as

Four second-sphere residues of Thermus thermophilus SG0.5JP17-16 laccase tune the catalysis by hydrogen-bonding networks

  • Huiping Liu
  • Yanyun Zhu
  • Xiaorong Yang
  • Ying Lin
Biotechnologically relevant enzymes and proteins


The multicopper oxidases catalyze 1-electron oxidation of four substrate molecules and concomitantly 4-electron reduction of dioxygen to water. The substrate loses the electrons at the type 1 copper (T1 Cu) site of the enzyme, while the dioxygen is reduced to water at the trinuclear copper center. A highly conserved Glu residue, which is at the dioxygen-entering channel, shuttles the proton to break the O-O bond of dioxygen. At the water-leaving channel, an Asp residue was found to be important in the protonation mechanism. In this study, laccase from Thermus thermophilus SG0.5JP17-16 (lacTT) was investigated to address how four second-sphere residues E356, E456, D106, and D423 affect the activity of the enzyme. Kinetic data indicate that catalytic activities of the enzyme are altered by site-directed mutagenesis on four second-sphere residues. The structural model of lacTT was generated by homology modeling. Structural and spectral data indicate that the E356 residue is situated at the substrate-binding site, responsible for the binding of the substrate and the geometry of the T1 Cu site by hydrogen-bonding networks; the E456 residue, located at the dioxygen-entering channel, plays a critical role in stabilizing the structure of all active copper centers and shuttling the proton to the trinuclear copper cluster (TNC) for the reductive reaction of dioxygen; the D106 and D423 residues are at the water-leaving channel, and they are important for the essential geometry of the TNC and the release of the water molecules. Altogether, this study contributes to the further understanding of the basic mechanism involving the oxidation of the substrate, electron transfer, and the reduction of dioxygen in lacTT.


Bacterial laccase Enzyme mechanism Enzyme kinetics Homology modeling Hydrogen-bonding network 



This study was supported by the National Natural Science Foundation of China (No. 41673074) and the Science and Technology Planning Project of Guangzhou City (No. 201607010073) to X Yang. All the authors are thankful for the financial support of the Science and Technology Planning Project of Guangzhou City (No. 201607010307).

Compliance with ethical standards

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

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

253_2018_8875_MOESM1_ESM.pdf (880 kb)
ESM 1 (PDF 879 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological EngineeringSouth China University of TechnologyGuangzhouPeople’s Republic of China
  2. 2.Guangdong Research Center of Industrial Enzyme and Green Manufacturing Technology, School of Biology and Biological EngineeringSouth China University of TechnologyGuangzhouPeople’s Republic of China

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