Domain structure and function of a-1,3-glucanase Agl-EK14 from the gram-negative bacterium Flavobacterium sp. EK-14
JOURNAL OF BIOSCIENCE AND BIOENGINEERING(2024)
摘要
The a-1,3-glucanase Agl-EK14 from Flavobacterium sp. EK-14 comprises a signal peptide (SP), a catalytic domain (CAT), a first immunoglobulin-like domain (Ig1), a second immunoglobulin-like domain (Ig2), a ricin B-like lectin domain (RicinB), and a carboxy-terminal domain (CTD). SP and CTD are predicted to be involved in extracellular secretion, while the roles of Ig1, Ig2, and RicinB are unclear. To clarify their roles, domain deletion enzymes Agl-EK14DRicinB, AglEK14DIg2RicinB, and Agl-EK14DIg1Ig2RicinB were constructed. The insoluble a-1,3-glucan hydrolytic, a-1,3-glucan binding, and fungal cell wall hydrolytic activities of the deletion enzymes were almost the same and lower than those of Agl-EK14. Kinetic analysis revealed that the Km values of the deletion enzymes were similar and uniformly higher than those of Agl-EK14. These results suggest that the deletion of RicinB causes a decline in binding and hydrolytic activity and increases the Km value. To confirm the role of RicinB, Ig1, Ig2, and RicinB were fused with green fluorescent protein (GFP). As a result, RicinB-fused GFP (GFP-RicinB) showed binding to insoluble a-1,3-glucan and Aspergillus oryzae cell walls, whereas Ig1- and Ig2-fused GFP did not. These results indicated that RicinB is involved in a1,3-glucan binding. The fusion protein GFP-Ig1Ig2RicinB was also constructed and GFP-Ig1Ig2RicinB showed strong binding to the cell wall of A. oryzae compared to GFP-RicinB. Gel filtration column chromatography suggested that the strong binding was due to GFP-Ig1Ig2RicinB loosely associated with itself. (c) 2024, The Society for Biotechnology, Japan. All rights are reserved, including those for text and data mining, AI training, and similar technologies.
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关键词
a-1,3-Glucanase,Glycoside hydrolase family 87,Flavobacterium,Fungal cell wall,3-Glucan binding domain
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