Structure, kinetic characterization and subcellular localization of the two ribulose 5-phosphate epimerase isoenzymes from Trypanosoma cruzi

Abstract

The enzyme of the pentose phosphate pathway (PPP) ribulose-5-phosphate-epimerase (RPE) is encoded by two genes present in the genome of Trypanosoma cruzi CL Brener clone: TcRPE1 and TcRPE2. Despite high sequence similarity at the amino acid residue level, the recombinant isoenzymes show a strikingly different kinetics. Whereas TcRPE2 follows a typical michaelian behavior, TcRPE1 shows a complex kinetic pattern, displaying a biphasic curve, suggesting the coexistence of -at least- two kinetically different molecular forms. Regarding the subcellular localization in epimastigotes, whereas TcRPE1 is a cytosolic enzyme, TcRPE2 is localized in glycosomes. To our knowledge, TcRPE2 is the first PPP isoenzyme that is exclusively localized in glycosomes. Over-expression of TcRPE1, but not of TcRPE2, significantly reduces the parasite doubling time in vitro, as compared with wild type epimastigotes. Both TcRPEs represent single domain proteins exhibiting the classical α/β TIM-barrel fold, as expected for enzymes with this activity. With regard to the architecture of the active site, all the important amino acid residues for catalysis -with the exception of M58- are also present in both TcRPEs models. The superimposition of the binding pocket of both isoenzyme models shows that they adopt essentially identical positions in the active site with a residue specific RMSD < 2Å, with the sole exception of S12, which displays a large deviation (residue specific RMSD: 11.07 Å). Studies on the quaternary arrangement of these isoenzymes reveal that both are present in a mixture of various oligomeric species made up of an even number of molecules, probably pointing to the dimer as their minimal functional unit. This multiplicity of oligomeric species has not been reported for any of the other RPEs studied so far and it might bear implications for the regulation of TcRPEs activity, although further investigation will be necessary to unravel the physiological significance of these structural findings.

CSGID Solves Structures and Identifies Phenotypes for Five Enzymes in Toxoplasma gondii

“…This pathway has been of interest in targeting various organisms, including P. falciparum and Trypanosoma cruzi (Barrett, 1997 ; Bozdech and Ginsburg, 2005 ; Igoillo-Esteve et al, 2007 ). The biology of RPE has proven to be of particular interest in T. cruzi (Gonzalez et al, 2017 ). Indeed, the structure of RPE had already been characterized in P. falciparum (Caruthers et al, 2005 ).…”

Enhanced Thermostability of D-Psicose 3-Epimerase from Clostridium bolteae through Rational Design and Engineering of New Disulfide Bridges

“…To determine the tertiary structure of C. bolteae DPEase, automatic homology modeling was performed via SWISS-MODEL using the Automated Mode method, and the known tertiary structure of A. tumefaciens DPEase was chosen as the template. The structural quality control analysis of the final 3D models chosen for Cb-DPEase was carried out using the Structural Analysis and Verification Server tools (SAVES; University of California, Los Angeles, CA, USA) [ 50 ].…”

Potential Drug Targets in the Pentose Phosphate Pathway of Trypanosomatids

“…In T. brucei RPE activity was detected in procyclics, but not in parasites isolated from mice [260]. Leishmania and T. cruzi encode for two isoenzymes that differ in the presence and absence of a PTS [271,272], and the first has recently been detected in L. donovani glycosomes [266]. Additionaly, its activity has been detected in L. mexicana promastigotes and was 3 fold higher than its competitor enzyme, RPIB [273].…”

“…In T. cruzi despite the high sequence similarity between the two isoenzymes the cytosolic TcRPE1 shows a specific activity 80-100-fold greater than the glycosomal isoenzyme TcRPE2 [271]. Furthermore, the subcellular localization of TcRPE2 is absolutely dependent on the presence of its targeting signal, since over-expression of a truncated mutant lacking the putative glycosomal targeting signal, not only remains in the cytosol, but also negatively affects parasite growth, extending its doubling time in vitro [271].…”