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Updated: Jul 17, 2026

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.
None:
Bacterial d-3-hydroxybutyrate dehydrogenases (HBDHs) catalyze the conversion between d-3-hydroxybutyrate and acetoacetate with NAD as the cofactor but not with NAD 2'-phosphate (NADP). However, HBDHs of the early-branched eukaryotic genus Trypanosoma utilize both NAD and NADP (T. brucei) or exclusively NADP (T. cruzi). Here we reveal that NADP specificity of T. cruzi HBDH arises from stabilization of the flexible β2αB loop by the 2'-phosphate interaction. Stabilization of this loop by a nearby C64Y mutation enables T. cruzi HBDH to use NAD in addition to NADP; thus, the Cys/Tyr residue is critical for determining cofactor specificity in trypanosomal HBDHs, suggesting that most trypanosomal HBDHs use both NAD and NADP except for T. cruzi HBDH. Furthermore, Arg42 within the β2αB loop interacts with the adenine ring of NADP by ideal CH-π interactions, while the R42F mutant switches to non-ideal π-π interactions, increasing kcat ∼10-fold and KM ∼40-fold. Collectively, we identified the β2αB loop stability and sequence as key determinants of NAD(P) co-factor specificity and kinetics in HBDHs.
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