The Glycosomal ATP‐Dependent Phosphofructokinase of Trypanosoma Brucei must have Evolved from an Ancestral Pyrophosphate‐Dependent Enzyme

Abstract

Trypanosoma brucei contains an ATP-dependent phosphofructokinase (PFK), located in its glycosomes, which are peroxisome-like organelles sequestering the majority of its glycolytic enzymes. In this paper, we report the cloning and sequencing of the single-copy gene encoding this enzyme. Its aminoacid sequence is more similar to pyrophosphate (PPJdependent PFKs than to other ATP-dependent PFKs. A phylogenetic analysis suggests that the enzyme must have been derived from a PP,-dependent ancestral PFK, which changed its phospho-donor specificity during evolution. The enzyme is no longer capable of using PP, as phospho substrate, nor can it catalyze the reverse reaction as PP,-PFKs generally can. Moreover, the presence of a high pyrophosphatase activity in the cell renders it unlikely that PP, can function as free-energy source in present-day trypanosomes. It remains to be determined which mutations were responsible for the change in phospho-substrate specificity of the trypanosomatid PFK. As a result of its particular evolutionary history, the iC brucei PFK shows many structural differences, even at the active site, when compared with other ATP-dependent PFKs. These differences offer great potential for the structure-based design of trypanocidal drugs.

Molecular and biochemical characterisation of Trypanosoma cruzi phosphofructokinase

“…4B); this result differed from the complex kinetics, with both negative and positive cooperativity, reported by Aguilar and Urbina (1986) at low F6P concentrations. However, our data agreed with the kinetic behaviour reported for this substrate in T. brucei (Michels et al 1997) and L. donovani (Lopez et al 2002), including slight cooperativity in the presence of AMP (not shown) with a Hill's coefficient of 1.4266.…”

“…Unlike human PFKs, the PFKs of kinetoplastids show the highest degree of sequence similarity with inorganic pyrophosphate (PP i )-dependent enzymes, despite being themselves ATP-dependent. In addition, effectors that modulate the activity of PFK in other organisms, such as ATP, citrate, fructose 2,6-bisphosphate and P i , have no effect in Trypanosoma brucei (Michels et al 1997) or Leishmania donovani (Lopez et al 2002). The many differences between the active site of the two classes of PFKs and the striking differences in ligand-binding properties between the human and parasite enzymes suggest great potential for structure-based design of drugs (Mertens 1991, Michels et al 1997).…”

“…In addition, effectors that modulate the activity of PFK in other organisms, such as ATP, citrate, fructose 2,6-bisphosphate and P i , have no effect in Trypanosoma brucei (Michels et al 1997) or Leishmania donovani (Lopez et al 2002). The many differences between the active site of the two classes of PFKs and the striking differences in ligand-binding properties between the human and parasite enzymes suggest great potential for structure-based design of drugs (Mertens 1991, Michels et al 1997). In a previous biochemical characterisation of Trypanosoma cruzi PFK (Aguilar & Urbina 1986), this enzyme was reported to have an apparent molecular mass of 17,000 and a complex kinetic pattern for its interaction with D-F6P.…”

Autophagy and Related processes in Trypanosomatids: Insights from Genomic and Bioinformatic Analyses

“…In contrast, ATG24 and VAC8 are proteins that interact with components of the ATG1-containing complex(es) that are present in trypanosomatids. PEX14, whose role in peroxisome biogenesis has been characterized in T. brucei 15 also has an ill-characterized role in induction of pexophagy. 39 The apparent absence of PEX3 reflects a likely case of gene displacement.…”

“…In the case of trypanosomatids, only two orthologues of yeast proteins involved in these pathways have been cloned and characterized, the peroxin PEX14 (refs. 13 and 14) and 6-phospho-1-fructokinase (PFK1); 15 however, these proteins were cloned with the objective to study their better known functions in glycosome biogenesis and glycolysis, respectively. Similarly, while the molecular and cellular biology of autophagy has been extensively studied, the structural biology of its participants is relatively neglected.…”