Allosteric Activation of Trypanosomatid Deoxyhypusine Synthase by a Catalytically Dead Paralog

Abstract

Background: Deoxyhypusine synthase (DHS) catalyzes the spermidine-dependent modification of translation factor eIF5A.Results: Trypanosomatid DHS activity is increased 3000-fold by heterotetramer formation with a catalytically dead paralog, and both gene products are essential for parasite growth.Conclusion: Trypanosomatid DHS is a complex between catalytically impaired and inactive DHS subunits.Significance: This activation mechanism uniquely evolved for two independent enzymes within the trypanosomatid polyamine pathway.

Deoxyhypusine Modification of Eukaryotic Translation Initiation Factor 5A (eIF5A) Is Essential for Trypanosoma brucei Growth and for Expression of Polyprolyl-containing Proteins

“…For quantitation of TbEIF5A mRNA levels in Figs. 2-4, RNA abundance was quantified using iTaq SYBR Green Supermix with ROX TM (Bio-Rad), and relative gene abundance was calculated by the ⌬⌬Ct method with TERT as the reference gene as described previously (26,36,37). For mRNA quantification shown in Fig.…”

“…Anti-TbeIF5A Antibody Production-Antibodies were raised in rabbits against purified recombinant TbeIF5A (Covance Inc.) that was purified as previously described (26).…”

“…DHS has been shown to be essential in all species where it has been studied including mammals and yeast (25) and more recently in the kinetoplastids T. brucei (26) and Leishmania (27). Furthermore, the kinetoplastids and Entamoeba encode two DHS genes, and it was shown for T. brucei that the two gene products (one catalytically active but impaired and the other catalytically inactive) associate to form a heterotetrameric enzyme, which is the functionally active form of the enzyme (26). This novel mechanism of enzyme activation by a catalytically inactive paralog was also observed for T. brucei S-adenosylmethionine decarboxylase, which is required for formation of the DHS substrate spermidine (28,29).…”

Genetic Validation of Trypanosoma brucei Glutathione Synthetase as an Essential Enzyme

“…The AdoMetDC regulatory protein prozyme is itself also an activator of AdoMetDC, as heterodimer formation with prozyme (an inactive paralog of AdoMetDC) increases enzymatic activity by 3 orders of magnitude. T. brucei deoxyhypusine synthase, an enzyme that uses spermidine as a substrate to modify an essential elongation factor (eIF5A), also requires activation by an inactive paralog (32). Despite these advances in understanding polyamine pathway regulation in T. brucei, key questions remain about how the glutathione branch of the pathway is regulated and how or if the polyamine and glutathione pathways show evidence for cross regulation.…”

“…The final yield was 0.8 mg of TbGS per liter of bacterial culture. Ulp1 was expressed and purified as previously described (31,32). All purification steps were performed at 4°C.…”