9K1K

Yeast Cytosine Deaminase mutant-M100H

Summary for 9K1K

• Entry DOI: 10.2210/pdb9k1k/pdb
• Descriptor: Cytosine deaminase, ZINC ION (3 entities in total)
• Functional Keywords: cytosine deaminase, antitumor protein
• Biological source: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (Baker's yeast)
• Total number of polymer chains: 2
• Total formula weight: 35335.57

Authors

Yung, K.W.Y.,Zheng, J.L.,Chan, M.K. (deposition date: 2024-10-16, release date: 2025-10-22, Last modification date: 2025-12-10)

Primary citation

Zheng, J.,Zhou, J.,Yung, K.W.Y.,Hu, Q.,Lee, M.M.,Chan, M.K.
An engineered yeast cytosine deaminase with improved catalytic activity and stability for macrophage-mediated enzyme/prodrug therapy.
Commun Biol, 8:1562-1562, 2025

PubMed Abstract: Utilization of yeast cytosine deaminase (yCD) to activate the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) at the target site is an attractive strategy for overcoming the narrow therapeutic index of 5-FU. Nevertheless, protein delivery of yCD is challenging in part due to its thermal instability. Herein, we have rationally engineered a mutant yCD by replacing Met100 situated at the active site entry with the bulkier histidine to hinder potential oxidation of the active site Cys91. The engineered yCD-Met100His exhibits significantly enhanced activity and thermal stability. yCD-M100H is then genetically fused to the crystal-forming protein Cry3Aa to generate Cry3Aa-yCD-M100H fusion crystals to facilitate the enzyme's uptake into macrophages. The resulting Cry3Aa-yCD-M100H-loaded macrophages exhibit excellent penetration into tumor spheroids and readily convert 5-FC to 5-FU leading to efficacious cancer cell killing. This study showcases a promising route for stabilizing yCD and the feasibility of enzyme-internalized macrophages to serve as tumor-specific enzyme/prodrug activators.

PubMed: 41233498
DOI: 10.1038/s42003-025-08931-x

Experimental method

X-RAY DIFFRACTION (1.439 Å)