Considerations regarding affinity determinants for aflatoxin B1 in binding cavity of fungal laccase based on in silico mutational and in vitro verification studies

Zhimin Zhou; Renkuan Li; Tzi Bun Ng; Fang Huang; Xiuyun Ye

doi:10.1016/j.ecoenv.2022.113412

Considerations regarding affinity determinants for aflatoxin B₁ in binding cavity of fungal laccase based on in silico mutational and in vitro verification studies

Ecotoxicol Environ Saf. 2022 Apr 1:234:113412. doi: 10.1016/j.ecoenv.2022.113412. Epub 2022 Mar 15.

Authors

Zhimin Zhou¹, Renkuan Li², Tzi Bun Ng³, Fang Huang², Xiuyun Ye⁴

Affiliations

¹ College of Chemical Engineering, Fuzhou University, Fuzhou 350116, China; The Key Laboratory of Marine Enzyme Engineering of Fujian Province, Fuzhou University, Fuzhou 350116, China; National Engineering Laboratory for High-efficient Enzyme Expression, Fuzhou 350116, China.
² The Key Laboratory of Marine Enzyme Engineering of Fujian Province, Fuzhou University, Fuzhou 350116, China; National Engineering Laboratory for High-efficient Enzyme Expression, Fuzhou 350116, China.
³ School of Life Sciences, Faculty of Science, The Chinese University of Hong Kong, Shatin, New Territories 999077, Hong Kong, China.
⁴ College of Chemical Engineering, Fuzhou University, Fuzhou 350116, China; The Key Laboratory of Marine Enzyme Engineering of Fujian Province, Fuzhou University, Fuzhou 350116, China; National Engineering Laboratory for High-efficient Enzyme Expression, Fuzhou 350116, China. Electronic address: xiuyunye@fzu.edu.cn.

PMID: 35304333
DOI: 10.1016/j.ecoenv.2022.113412

Abstract

Laccase, a multicopper oxidase, is well known for its industrial potentials to remove environmental pollutants due to its low substrate specificity to oxidize phenols and thus catalytic versatility. Many efforts focused on the metabolic mechanism, yet to decipher the structural determinants responsible for the differentiation between substrates. Aflatoxin B₁ (AFB₁), a new substrate for laccase, is a mycotoxin with a formidable environmental threat to public health and food safety. In the present study, we combined biochemical, in silico mutational and molecular-docking data to gain an insight to the function of key residues in the active cavity close to the T1 copper site in a characterized recombinant laccase from Cerrena unicolor (rCuL). Kinetic data for computer-assisted virtual mutants established the binding affinity of hydrogen bonds and residues (Asn336, Asp207, Val391, and Thr165) in rCuL to AFB₁. The augmented binding affinity to AFB₁ may be related to the conformational rearrangements of the laccase and its ability to hydrogen-bond with the substrate. Furthermore, the optimal pH and temperature for rCuL and variants mediated AFB₁ degradation may depend on their pH stability and thermostability. Our findings reinforce the importance of the structure-function relationship of fungal laccases in degrading AFB₁, providing mechanistic guidance for future biocatalyst and bioengineering applications.

Keywords: Affinity; Aflatoxin B(1); Degradation; Laccase; Mutation.