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Updated: Aug 14, 2026
10:27
Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta
Published on: December 5, 2019
Catalytic loop closure governs substrate selectivity in the rhizocticin biosynthetic ligases RhiM and RhiC
Mizuki Sakai1, Ryosuke Masuda2, Yu Hirano3
1Quantum Life Science Course, Graduate School of Science and Engineering, Chiba University, Inage, Chiba 263-8522, Japan; Institute for Quantum Life Science, National Institutes for Quantum Science and Technology, Inage, Chiba 263-8555, Japan.
The Journal of Biological Chemistry
|August 12, 2026
Summary
RhiM and RhiC enzymes, crucial for antifungal peptide rhizocticin, were structurally analyzed. This revealed how their catalytic loops reconfigure to bind substrates and facilitate antifungal peptide biosynthesis.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- RhiM and RhiC are ATP-dependent ligases essential for rhizocticin antifungal peptide biosynthesis.
- Previous studies clarified their functions but lacked structural insights into catalytic mechanisms and substrate selectivity.
Purpose of the Study:
- To elucidate the molecular basis of RhiM and RhiC catalytic mechanisms and substrate selectivity.
- To obtain structural and biochemical evidence for the proposed acyl phosphate intermediate.
Main Methods:
- X-ray crystallography was employed to determine crystal structures of RhiM and RhiC.
- Structures were obtained for nucleotide-bound, substrate-bound, and intermediate-analog-bound forms, alongside biochemical and mutational analyses.
Main Results:
- Crystal structures revealed nucleotide-bound RhiM and RhiC, and substrate/intermediate-analog-bound RhiC.
- Structural data provided evidence for an acyl phosphate intermediate mechanism in RhiC.
- Conformational changes in catalytic loops were identified as critical for substrate binding, positioning, and enzyme activity.
Conclusions:
- Catalytic loop rearrangements are essential for substrate pocket formation and precise substrate/intermediate positioning.
- The study provides structural evidence supporting the acyl phosphate intermediate mechanism.
- These findings illuminate the molecular mechanisms governing RhiM and RhiC activity and substrate selectivity.
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