Identification and characterization of a novel Ribose 5-phosphate isomerase B from Leishmania donovani

Abstract

Leishmaniasis is a group of tropical diseases caused by protozoan parasites of the genus Leishmania. Due to the emergence of resistance to the available antileishmanial drugs there is an immediate need to identify molecular targets on which to base future treatment strategies. Ribose 5-phosphate isomerase (Rpi; EC 5.3.1.6) is a key enzyme of the pentose phosphate pathway (PPP) which catalyses the reversible aldose-ketose isomerization between Ribose 5-phosphate (R5P) and Ribulose 5-phosphate (Ru5P). It exists in two isoforms A and B. These two are completely unrelated enzymes catalyzing the same reaction. Analysis of the Leishmania infantum genome revealed that though the RpiB gene is present, RpiA homologs are completely absent. An absence of RpiBs in the genomes of higher animals makes this enzyme a possible target for the chemotherapy of Leishmaniasis. In this paper, we report for the first time the presence of B isoform of the Rpi enzyme in Leishmania donovani (LdRpiB) by cloning and molecular characterization of the enzyme. An amplified L. donovani RpiB gene is 519bp and encodes for a putative 172 amino acid protein with a molecular mass of ∼19kDa. An ∼19kDa protein with poly-His tag at the C-terminal end was obtained by heterologous expression of LdRpiB in Escherichia coli. The recombinant form of RpiB was obtained in soluble and active form. The LdRpiB exists as a dimer of dimers i.e. the tetramer form. The polyclonal antibody against Trypanosoma cruzi RpiB could detect a band of ∼19kDa with the purified recombinant RpiB as well as native RpiB from the L. donovani promastigotes. Recombinant RpiB obeys the classical Michaelis–Menten kinetics utilizing R5P as the substrate with a Km value of 2.4±0.6mM and Kcat value of 30±5.2s−1. Our study confirms the presence of Ribose 5-phosphate isomerase B in L. donovani and provides functional characterization of RpiB for further validating it as a potential drug target.