Document Type

Article

Publication Date

10-1-2026

Comments

This article is the author’s final published version in Journal of Molecular Biology, Volume 438, Issue 19, 2026, Article number 169946.

The published version is available at https://doi.org/10.1016/j.jmb.2026.169946. Copyright © 2026 The Authors.

 

Abstract

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.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial 4.0 License

Additional Files
Appendix A. Supplementary material.pdf (23875 kB)

PubMed ID

42456964

Language

English

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Available for download on Thursday, October 01, 2026

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