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A Unique Insertion Loop Facilitates Tight NAD+ Binding in Nicotinoprotein: Insights from In Vitro Loop Engineering
Houcheng Xue1, Takumi Yanase1, Junko Okuda-Shimazaki1
1Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
International Journal of Molecular Sciences
|March 14, 2026
Summary
The unique insertion loop in nicotinoproteins is essential for tight Nicotinamide Adenine Dinucleotide (NAD+) binding, but it hinders NAD+ entry into the active site, impacting enzyme activity.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Nicotinoproteins are NAD+-dependent dehydrogenases with tight NAD+ binding.
- The role of the unique insertion loop in nicotinoproteins remains unclear.
Purpose of the Study:
- Investigate the function of the insertion loop in nicotinoproteins.
- Determine the loop's necessity and sufficiency for NAD+ tight binding.
Main Methods:
- Enzyme engineering: created insertion loop-deleted mutant (CADh Δ39-49) and loop-swapped mutants.
- Biochemical assays: measured NAD+ binding and dehydrogenase activity.
- Molecular docking simulations: analyzed NAD+ binding affinity.
Main Results:
- CADh Δ39-49 lost NAD+ tight binding and free NAD+ utilization.
- Mutants showed altered NAD+ binding kinetics (higher K_M and K_D).
- Docking simulations indicated stronger NAD+ binding in mutants, suggesting interference with NAD+ entry.
Conclusions:
- The insertion loop is necessary for tight NAD+ binding in SDR nicotinoproteins.
- The loop impedes NAD+ access to the active site, creating a favorable binding environment.
- Findings offer insights for engineering SDR enzymes with modified NAD+ binding properties.
Keywords:
NAD+-dependent dehydrogenasenicotinoproteinprotein engineeringshort-chain dehydrogenase/reductaseMore Related Videos
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