AtaA as a Self-Immobilizing Scaffold for Enzymatically Active Surfaces through Oxidative Tyrosine Coupling
- 1Nagahama Institute of Bio-Science and Technology
Abstract
Protein nanofibers are hold great promise as genetically programmable scaffolds for constructing functional material interfaces. AtaA, a giant trimeric autotransporter adhesin from Acinetobacter sp. Tol 5, forms an elongated architecture from the bacterial cell surface, enabling the cells to adhere directly to a wide variety of abiotic surfaces. However, the potential use of AtaA as a scaffold with additional biological functions remains largely unexplored. In this study, we investigated the use of AtaA as a self-immobilizing scaffold for constructing enzymatically active surfaces via horseradish peroxidase (HRP)-catalyzed tyrosine coupling. We prepared a recombinant AtaA nanofiber fragment (NhNs-AtaA) and its derivative containing two tyrosine-containing loop structures (YL-NhNs-AtaA). Both recombinant proteins retained their adhesive properties. Treatment with HRP and H2O2 generated high-molecular-weight products from both proteins, and dityrosine fluorescence emission indicated the formation of tyrosine-derived covalent cross-links. Y-Loop insertion further increased the consumption of monomeric AtaA and promoted the formation of high-molecular-weight products. The removal of arginine, which was included during purification, further increased the loss of monomeric AtaA in both constructs, indicating that intermolecular protein association influenced productive AtaA cross-linking. NhNs-AtaA and YL-NhNs-AtaA were combined with Y-tagged bacterial alkaline phosphatase (BAP-Y). HRP-mediated coupling generated high-molecular-weight products consistent with the cross-linked AtaA-BAP-Y conjugates and enabled the retention of BAP activity on AtaA-coated surfaces without prior chemical modification. Although Y-Loop insertion enhanced polymerization in solution, it reduced AtaA adhesion and consequently lowered surface-bound phosphatase activity, revealing a trade-off between cross-linking reactivity and native adhesive function. These findings establish AtaA as a self-immobilizing protein nanofiber scaffold for constructing enzymatically active surfaces, and suggest the potential extension of this strategy to other AtaA-based interfaces.
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BibTeX
@misc{yokoe2026ataa,
title = {{AtaA as a Self-Immobilizing Scaffold for Enzymatically Active Surfaces through Oxidative Tyrosine Coupling}},
author = {Y. Yokoe and S. Yoshimoto and K. Miniamihata and R. Sugimoto and A. Nagatani and A. Nara and N. Kamiya and K. Hori and M. Ishikawa},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.30.755837},
url = {https://www.biorxiv.org/content/10.64898/2026.09.30.755837v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - AtaA as a Self-Immobilizing Scaffold for Enzymatically Active Surfaces through Oxidative Tyrosine Coupling
AU - Y. Yokoe
AU - S. Yoshimoto
AU - K. Miniamihata
AU - R. Sugimoto
AU - A. Nagatani
AU - A. Nara
AU - N. Kamiya
AU - K. Hori
AU - M. Ishikawa
PY - 2026
DA - 2026/09/30
PB - bioRxiv
DO - 10.64898/2026.09.30.755837
UR - https://www.biorxiv.org/content/10.64898/2026.09.30.755837v1
AB - Protein nanofibers are hold great promise as genetically programmable scaffolds for constructing functional material interfaces. AtaA, a giant trimeric autotransporter adhesin from Acinetobacter sp. Tol 5, forms an elongated architecture from the bacterial cell surface, enabling the cells to adhere directly to a wide variety of abiotic surfaces. However, the potential use of AtaA as a scaffold with additional biological functions remains largely unexplored. In this study, we investigated the use of AtaA as a self-immobilizing scaffold for constructing enzymatically active surfaces via horseradish peroxidase (HRP)-catalyzed tyrosine coupling. We prepared a recombinant AtaA nanofiber fragment (NhNs-AtaA) and its derivative containing two tyrosine-containing loop structures (YL-NhNs-AtaA). Both recombinant proteins retained their adhesive properties. Treatment with HRP and H2O2 generated high-molecular-weight products from both proteins, and dityrosine fluorescence emission indicated the formation of tyrosine-derived covalent cross-links. Y-Loop insertion further increased the consumption of monomeric AtaA and promoted the formation of high-molecular-weight products. The removal of arginine, which was included during purification, further increased the loss of monomeric AtaA in both constructs, indicating that intermolecular protein association influenced productive AtaA cross-linking. NhNs-AtaA and YL-NhNs-AtaA were combined with Y-tagged bacterial alkaline phosphatase (BAP-Y). HRP-mediated coupling generated high-molecular-weight products consistent with the cross-linked AtaA-BAP-Y conjugates and enabled the retention of BAP activity on AtaA-coated surfaces without prior chemical modification. Although Y-Loop insertion enhanced polymerization in solution, it reduced AtaA adhesion and consequently lowered surface-bound phosphatase activity, revealing a trade-off between cross-linking reactivity and native adhesive function. These findings establish AtaA as a self-immobilizing protein nanofiber scaffold for constructing enzymatically active surfaces, and suggest the potential extension of this strategy to other AtaA-based interfaces.
ER -