Premium
Characterization of an unusual Trypanosoma brucei beta‐hydroxybutyrate dehydrogenase
Author(s) -
Shah Tina D,
Hickey Meghan,
Palenchar Jennifer B
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.913.6
Subject(s) - trypanosoma brucei , nad+ kinase , enzyme , cofactor , biochemistry , dehydrogenase , biology , alcohol dehydrogenase , microbiology and biotechnology , gene
We report the characterization of an unusual β‐hydroxybutyrate dehydrogenase ( Tb βHBDH) from Trypanosoma brucei , the unicellular eukaryotic parasite responsible for causing African Sleeping Sickness. βHBDH catalyzes the reversible NADH‐dependent conversion of acetoacetate to hydroxybutyrate. Unlike its eukaryotic homologs, Tb βHBDH does not appear to be a membrane protein and has greater sequence similarity to its soluble bacterial counterparts. The enzyme was cloned from T. brucei genomic DNA, and active, recombinant enzyme was purified from E. coli . Kinetic studies were carried out to determine the K M , V max , and K i values for substrates and inhibitors. Cofactor preference was examined for Tb βHBDH and it was found that Tb βHBDH utilizes both NAD(H) and NADP(H) equally, distinguishing the parasite enzyme from other characterized βHBDHs. Furthermore, the enzyme appears to bind NAD(P) + in a cooperative fashion. Through size exclusion chromatography, the apparent molecular weight of Tb βHBDH is 96.5 kDa. RNA interference (RNAi) studies were carried out to begin to determine the in vivo role of Tb βHBDH. Upon knockdown of Tb βHBDH, verified through Western blot analysis, a noticeable reduction in parasite growth was observed; suggesting βHBDH has an important physiological role in T. brucei. This work is funded by the Research Corporation for Science Advancement.