Orthogonal Enzymatic Conjugation Reactions Create Chitin Binding Domain Grafted Chitinase Polymers with Enhanced Antifungal Activity

Abstract

Enzymatic reaction offers site-specific conjugation of protein units to form protein conjugates or protein polymers with intrinsic functions. Herein, we report horseradish peroxidase (HRP)-and microbial transglutaminase (MTG)-catalyzed orthogonal conjugation reactions to create antifungal protein polymers composed of Pteris ryukyuensis chitinase-A (ChiA) and its two domains, catalytic domain, CatD, and chitin-binding domain, LysM 2 . We engineered the ChiA and CatD by introducing a peptide tag containing tyrosine (Y-tag) at N-termini and a peptide tag containing lysine and tyrosine (KY-tag) at C-termini to construct Y-ChiA-KY and Y-CatD-KY. Also, LysM 2 with Y-tag and KY-tag (Y-LysM 2 -KY) or with a glutamine-containing peptide tag (Q-tag) (LysM 2 -Q) were constructed. The proteins with Y-tag and KY-tag were efficiently polymerized by HRP reaction through the formation of dityrosine bonds at the tyrosine residues in the peptide tags. The Y-CatD-KY polymer was further treated by MTG to orthogonally graft LysM 2 -Q to the KY-tag via isopeptide formation between the side chains of the glutamine and lysine residues in the peptide tags to form LysM 2 -grafted CatD polymer. The LysM 2 -grafted CatD polymer exhibited significantly higher antifungal activity than the homopolymer of Y-ChiA-KY and the random copolymer of Y-CatD-KY and Y-LysM 2 -KY, demonstrating that the structural differences of artificial chitinase polymers have a significant impact on the antifungal activity. This strategy of polymerization and grafting reaction of protein can contribute to the further research and development of functional protein polymers for specific applications in various fields in biotechnology.

Enhancement of the Antifungal Activity of Chitinase by Palmitoylation and the Synergy of Palmitoylated Chitinase with Amphotericin B

“…However, the antifungal activity of PrChiA and its domains is not sufficient for use as an antifungal agent, and they need to be administered at high concentrations to exert antifungal activity. To improve the antifungal activity of chitinase, we previously prepared a LysM-grafted CatD polymer, which had increased activity . Briefly, the CatD was genetically engineered, and peptide tags containing a tyrosine residue (Y-tag) and tyrosine and lysine residues (KY-tag) were introduced to the N- and C-termini, respectively.…”

Chemical and Enzymatic Methods for Post-Translational Protein–Protein Conjugation

“…The C-terminal Y-tag G 4 Y has also been targeted with other oxidizing enzymes for protein–protein conjugation . Treatment with enzymes such as laccase or horseradish peroxidase in conjunction with H 2 O 2 can lead to the formation of protein–protein heterodimers including an IgG partner, although higher order oligomeric species have also been observed. − …”

Designed protein multimerization and polymerization for functionalization of proteins

“…An impressive study of the HRP- and MTG-catalyzed formation of polymeric chitinase was reported by Minamihata et al ( 2021 ) who engineered a chitinase from P. ryukuensis (ChiA) by removing the chitin binding domain (LysM 2 ) and modifying the catalytic domain (CatD) of chitinase by adding Y-and KY-tags at the N-and C-termini of CatD, respectively. They also engineered the wild-type ChiA by adding Y-and KY-tags, similar to the CatD, to construct Y-ChiA-KY and used it as a control.…”

“…F Results of inhibition assay of WT- ChiA, CatD/LysM 2 copolymer, and LysM 2 G/CatD polymer against T. viride . Reproduced from Minamihata et al ( 2021 ). Copyright 2021 with permission from the American Chemical Society (ACS) …”

“… Reproduced from Minamihata et al ( 2021 ). Copyright 2021 with permission from the American Chemical Society (ACS) …”