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Updated: Jul 17, 2026
08:44
Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei
Published on: January 22, 2019
The β2αB loop determines NAD(P) cofactor specificity and kinetics in trypanosomal D-3-hydroxybutyrate dehydrogenases
Hideharu Hashimoto1, Ian H Mawn1, William Escobar-Arrilaga2
1Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107, USA.
Journal of Molecular Biology
|July 15, 2026
Summary
Cofactor specificity in d-3-hydroxybutyrate dehydrogenases (HBDHs) from Trypanosoma is determined by the β2αB loop. Mutations reveal how this loop
Area of Science:
- Biochemistry
- Enzymology
- Parasitology
Background:
- Bacterial d-3-hydroxybutyrate dehydrogenases (HBDHs) typically use NAD as a cofactor.
- Trypanosoma HBDHs exhibit varied cofactor preferences, utilizing NADP or both NAD and NADP.
- Understanding cofactor specificity is crucial for enzyme mechanism elucidation.
Purpose of the Study:
- To investigate the molecular basis of NADP specificity in Trypanosoma cruzi HBDH.
- To identify key residues and structural features governing cofactor preference and enzyme kinetics.
- To explore the evolutionary implications of cofactor utilization in trypanosomal HBDHs.
Main Methods:
- Site-directed mutagenesis of T. cruzi HBDH (C64Y and R42F mutations).
- Enzyme kinetics assays to determine catalytic efficiency (kcat) and Michaelis constant (KM).
- Structural analysis focusing on the β2αB loop and cofactor interactions.
Main Results:
- NADP specificity in T. cruzi HBDH is linked to the stabilization of the flexible β2αB loop by the 2'-phosphate.
- A C64Y mutation allows T. cruzi HBDH to utilize NAD, highlighting the Cys/Tyr residue's role in specificity.
- The R42F mutation alters cofactor interactions, significantly impacting enzyme kinetics (kcat and KM).
Conclusions:
- The stability and sequence of the β2αB loop are critical determinants of NAD(P) cofactor specificity in HBDHs.
- Cofactor specificity in trypanosomal HBDHs is modulated by specific amino acid residues within the β2αB loop.
- These findings provide insights into the evolution of enzyme cofactor usage in eukaryotes.
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