Cloning, sequencing and expression of ribonucleotide reductase R2 from Trypanosoma brucei

Abstract

As a first step towards the understanding of the trypanosomal synthesis of deoxyribonucleotides we describe the cloning of the gene of ribonucleotide reductase subunit R2 from 72 brucei hrucei and its overexpression in E. coli. Materials and methods DNA and RNA preparation2;4 l08 long slender 72 brucei cells were suspended in 10 mM Tris-HC1, 250 mM NaCI, 0.5% Nonidet P-40, pH 8.0. After 5 rain incubation on ice the suspension was centrifuged and the cell pellet was homogenized in 200 lal 10 mM Tris-HC1, 10 mM NaC1, 10 mM EDTA, 0.5% SDS, pH 8.0. 50 lag proteinase K were added and the mixture was incubated for 1 h at 37°C. DNA was purified by phenol extraction.RNA was isolated from bloodstream long slender and short stumpy as well as procyclic stages of 72 brucei using the RNA Easy Kit (Qiagen) according to the instructions of the manufacturer.

Preliminary Structure–Activity Relationship Study of the MMV Pathogen Box Compound MMV675968 (2,4-Diaminoquinazoline) Unveils Novel Inhibitors of Trypanosoma brucei brucei

“…In addition, Ayayi et al [ 55 ] investigated the iron dependence of oxidase alternative and terminal trypanosomes (AOT) by chelating iron using o-phenanthroline, which resulted in strong inhibition of this enzyme. Ribonucleotide reductase is another iron-dependent enzyme that catalyzes the reduction of ribonucleotides to deoxyribonucleotides needed for DNA synthesis [ 56 , 57 ]. Therefore, iron deprivation of parasites by compound 7 might induce a loss of viability of these vital enzymes, thereby resulting in a rapid decrease in DNA synthesis, increased oxidative stress and cessation of electron transfer to the AOT enzyme, thus contributing to the death of the parasite.…”

Iron Uptake Controls Trypanosoma cruzi Metabolic Shift and Cell Proliferation

“…The concentration of free Fe in the environment ranges between 10 −9 and 10 −18 M, which is lower than the concentration necessary for microbial development [8]. Fe is required for DNA synthesis [9,10], energy generation [11], and oxidative stress in trypanosomes [12]. Furthermore, mammalian hosts sequester free Fe into proteins such as transferrin and lactoferrin [13][14][15], resulting in a free Fe concentration in serum of roughly 10 −24 M [13][14][15].…”

Rudimentary Structure-Activity-Relationship study of the MMV Pathogen Box compound MMV675968 (2,4-diaminoquinazoline) unveils novel inhibitors of Trypanosoma brucei brucei DHFR enzyme

“…In addition, Ayayi et al [67] investigated the iron dependence of oxidase alternative and terminal trypanosomes (AOT) by chelating iron using o-phenanthroline, which resulted in strong inhibition of this enzyme. Ribonucleotide reductase is another iron-dependent enzyme that catalyzes the reduction of ribonucleotides to deoxyribonucleotides needed for DNA synthesis [68, 69]. Therefore, iron deprivation of parasites by compound 7 might induce a loss of viability of these vital enzymes, thereby resulting in a rapid decrease in DNA synthesis, increased oxidative stress and cessation of electron transfer to the AOT enzyme, thus contributing to the death of the parasite.…”