The characterization of a unique Trypanosoma brucei β-hydroxybutyrate dehydrogenase

A β-hydroxybutyrate dehydrogenase ortholog in Trypanosoma brucei was identified and characterized. The trypanosome enzyme has unique kinetic properties among the β-hydroxybutyrate dehydrogenases characterized to date. [Display omitted] ► Trypanosoma brucei contains an active β-hydroxybutyrate dehydrogenase ortholog. ► By RNA interference, the enzyme is important for procyclic cell growth. ► Both NADP(H) and NAD(H) are utilized by the enzyme. ► The oxidized form of the cofactor is bound cooperatively. A putative β-hydroxybutyrate dehydrogenase (βHBDH) ortholog was identified in Trypanosoma brucei, the unicellular eukaryotic parasite responsible for causing African Sleeping Sickness. The trypanosome enzyme has greater sequence similarity to bacterial sources of soluble βHBDH than to membrane-bound Type I βHBDH found in higher eukaryotes. The βHBDH gene was cloned from T. brucei genomic DNA and active, recombinant His-tagged enzyme (His10-TbβHBDH) was purified to approximate homogeneity from E. coli. βHBDH catalyzes the reversible NADH-dependent conversion of acetoacetate to d-3-hydroxybutyrate. In the direction of d-3-hydroxybutyrate formation, His10-TbβHBDH has a kcat value of 0.19s−1 and a KM value of 0.69mM for acetoacetate. In the direction of acetoacetate formation, His10-TbβHBDH has a kcat value of 11.2s−1 and a KM value of 0.65mM for d-3-hydroxybutyrate. Cofactor preference was examined and His10-TbβHBDH utilizes both NAD(H) and NADP(H) almost equivalently, distinguishing the parasite enzyme from other characterized βHBDHs. Furthermore, His10-TbβHBDH binds NAD(P)+ in a cooperative fashion, another unique characteristic of trypanosome βHBDH. The apparent native molecular weight of recombinant His10-TbβHBDH is 112kDa, corresponding to tetramer, as determined through size exclusion chromatography. RNA interference studies in procyclic trypanosomes were carried out to evaluate the importance of TbβHBDH in vivo. Upon knockdown of TbβHBDH, a small reduction in parasite growth was observed suggesting βHBDH has an important physiological role in T. brucei.