Molecular Characterization of the First Two Enzymes of the Pentose-phosphate Pathway of Trypanosoma brucei

Duffieux, Francis; Roy, Joris Van; Michels, Paul A.M.; Opperdoes, Frederik

doi:10.1074/jbc.m004266200

Cited by 79 publications

(59 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…It has been proposed that the role of the PGL enzyme in vivo is rapid and effective hydrolysis of lactones, preventing ester-and amide-forming reactions involving, e.g., basic amino acids, such as arginine and lysine (6). In the light of our results, there is complete agreement with the idea that the role of the PGL enzyme is to facilitate the flow of metabolic intermediates through the PP shunt.…”

Section: Figsupporting

confidence: 81%

Suppressing Posttranslational Gluconoylation of Heterologous Proteins by Metabolic Engineering of Escherichia coli

Aon¹,

Caimi²,

Taylor³

et al. 2008

Appl Environ Microbiol

View full text Add to dashboard Cite

Minimization of chemical modifications during the production of proteins for pharmaceutical and medical applications is of fundamental and practical importance. The gluconoylation of heterologously expressed protein which is observed in Escherichia coli BL21(DE3) constitutes one such undesired posttranslational modification. We postulated that formation of gluconoylated/phosphogluconoylated products of heterologous proteins is caused by the accumulation of 6-phosphogluconolactone due to the absence of phosphogluconolactonase (PGL) in the pentose phosphate pathway. The results obtained demonstrate that overexpression of a heterologous PGL in BL21(DE3) suppresses the formation of the gluconoylated adducts in the therapeutic proteins studied. When this E. coli strain was grown in high-cell-density fed-batch cultures with an extra copy of the pgl gene, we found that the biomass yield and specific productivity of a heterologous 18-kDa protein increased simultaneously by 50 and 60%, respectively. The higher level of PGL expression allowed E. coli strain BL21(DE3) to satisfy the extra demand for precursors, as well as the energy requirements, in order to replicate plasmid DNA and express heterologous genes, as metabolic flux analysis showed by the higher precursor and NADPH fluxes through the oxidative branch of the pentose phosphate shunt. This work shows that E. coli strain BL21(DE3) can be used as a host to produce three different proteins, a heterodimer of liver X receptors, elongin C, and an 18-kDa protein. This is the first report describing a novel and general strategy for suppressing this nonenzymatic modification by metabolic pathway engineering.

show abstract

Section: Figsupporting

confidence: 81%

Suppressing Posttranslational Gluconoylation of Heterologous Proteins by Metabolic Engineering of Escherichia coli

Aon¹,

Caimi²,

Taylor³

et al. 2008

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…We expressed the T. cruzi G6PDH in both, its long and short forms (Igoillo-Esteve and Cazzulo 2006) and compared both recombinant enzymes with the native one. On the other hand, the T. brucei G6PDH was only expressed in its short form, starting from the second Met residue (Duffieux et al 2000). Due to the low levels of expression of the native G6PDH in epimastigotes from T. cruzi, added to the instability of the enzyme, we were unable to purify it to homogeneity by standard procedures.…”

Section: Glucose 6-phosphate Dehydrogenasementioning

confidence: 99%

“…A comparison of its sequence with that of the enzyme from T. brucei (Fig. 3) indicates that they are 62% identical, and both predict a possible PTS 1 glycosomal targeting signal, placed at the C-terminus in the case of T. brucei and internal in the T. cruzi 6-PGL (P. Beluardi and J.J. Cazzulo, unpublished results, Duffieux et al 2000). The T. cruzi 6-PGL behaves as a monomeric enzyme, with a molecular mass of 29 kDa.…”

Section: -Phosphogluconolactonasementioning

confidence: 99%

“…Nevertheless, its biological relevance is not very clear until now; we suggest that in T. cruzi a G6PDH fraction associated with the Golgi complex would be indirectly protecting the lipid peroxidation in the organelle membranes via the microsomal Tc-GPXII (Wilkinson et al 2002b). The G6PDH from procyclic and bloodstream trypomastigotes from T. brucei has a dual subcellular localization, cytosolic and glycosomal (Heise and Opperdoes 1999), despite the apparent lack of a PTS signal (Duffieux et al 2000).…”

Section: Glucose 6-phosphate Dehydrogenasementioning

confidence: 99%

See 1 more Smart Citation

The pentose phosphate pathway in Trypanosoma cruzi: a potential target for the chemotherapy of Chagas disease

Igoillo-Esteve

Maugeri

Stern

et al. 2007

An. Acad. Bras. Ciênc.

View full text Add to dashboard Cite

Trypanosoma cruzi is highly sensitive to oxidative stress caused by reactive oxygen species. Trypanothione, the parasite's major protection against oxidative stress, is kept reduced by trypanothione reductase, using NADPH; the major source of the reduced coenzyme seems to be the pentose phosphate pathway. Its seven enzymes are present in the four major stages in the parasite's biological cycle; we have cloned and expressed them in Escherichia coli as active proteins. Glucose 6-phosphate dehydrogenase, which controls glucose flux through the pathway by its response to the NADP/NADPH ratio, is encoded by a number of genes per haploid genome, and is induced up to 46-fold by hydrogen peroxide in metacyclic trypomastigotes. The genes encoding 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, transaldolase and transketolase are present in the CL Brener clone as a single copy per haploid genome. 6-phosphogluconate dehydrogenase is very unstable, but was stabilized introducing two salt bridges by site-directed mutagenesis. Ribose-5-phosphate isomerase belongs to Type B; genes encoding Type A enzymes, present in mammals, are absent. Ribulose-5-phosphate epimerase is encoded by two genes. The enzymes of the pathway have a major cytosolic component, although several of them have a secondary glycosomal localization, and also minor localizations in other organelles.

show abstract

“…Nos tripanossomatídeos, as enzimas da via PPP ocorrem tanto no glicossomo quanto no citossol (HEISE et al, 1999;DUFFIEUX et al, 2000). Esta característica poderia ser explicada pelas seguintes evidências: (i) a via glicolítica e da PPP podem trocar intermediários para ajustar as necessidades celulares de ATP; (ii) o produto final, ribose-5-fosfato, é convertido em 5-ribosil-1-pirofosfato que é utilizado na síntese de purinas e pirimidinas, processo que ocorre nos glicossomos; (iii) as enzimas envolvidas na defesa contra EROs são encontradas em ambos compartimentos (WILKINSON et al, 2002a).…”

Section: Via Das Pentoses Fosfato (Ppp)unclassified

NADH desidrogenase mitocondrial de Trypanosoma cruzi: subunidade 7 para diagnóstico diferencial de isolados humanos e análise funcional.

Martínez¹

View full text Add to dashboard Cite

Molecular Characterization of the First Two Enzymes of the Pentose-phosphate Pathway of Trypanosoma brucei

Cited by 79 publications

References 49 publications

Suppressing Posttranslational Gluconoylation of Heterologous Proteins by Metabolic Engineering of Escherichia coli

Suppressing Posttranslational Gluconoylation of Heterologous Proteins by Metabolic Engineering of Escherichia coli

The pentose phosphate pathway in Trypanosoma cruzi: a potential target for the chemotherapy of Chagas disease

NADH desidrogenase mitocondrial de Trypanosoma cruzi: subunidade 7 para diagnóstico diferencial de isolados humanos e análise funcional.

Contact Info

Product

Resources

About