Boniface M Mailu

Seattle Institute for Biomedical and Clinical Research, Seattle, Washington, United States

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Publications (9)24.09 Total impact

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    ABSTRACT: The malaria parasite Plasmodium falciparum and related organisms possess a relict plastid known as the apicoplast. Apicoplast protein synthesis is a validated drug target in malaria because antibiotics that inhibit translation in prokaryotes also inhibit apicoplast protein synthesis and are sometimes used for malaria prophylaxis or treatment. We identified components of an indirect aminoacylation pathway for Gln-tRNAGln biosynthesis in Plasmodium that we hypothesized would be essential for apicoplast protein synthesis. Here we report our characterization of the first enzyme in this pathway, the apicoplast glutamyl-tRNA synthetase (GluRS). We expressed the recombinant P. falciparum enzyme in E. coli, showed that it is non-discriminating because it glutamylates both apicoplast tRNAGlu and tRNAGln, determined its kinetic parameters, and demonstrated its inhibition by a known bacterial GluRS inhibitor. We also localized the P. berghei ortholog to the apicoplast in blood stage parasites but could not delete the PbGluRS gene. These data show that Gln-tRNAGln biosynthesis in the Plasmodium apicoplast proceeds via an essential indirect aminoacylation pathway that is reminiscent of bacteria and plastids.
    Journal of Biological Chemistry 09/2013; · 4.65 Impact Factor
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    ABSTRACT: The survival of malaria parasites in human RBCs (red blood cells) depends on the pentose phosphate pathway, both in Plasmodium falciparum and its human host. G6PD (glucose-6-phosphate dehydrogenase) deficiency, the most common human enzyme deficiency, leads to a lack of NADPH in erythrocytes, and protects from malaria. In P. falciparum, G6PD is combined with the second enzyme of the pentose phosphate pathway to create a unique bifunctional enzyme named GluPho (glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase). In the present paper, we report for the first time the cloning, heterologous overexpression, purification and kinetic characterization of both enzymatic activities of full-length PfGluPho (P. falciparum GluPho), and demonstrate striking structural and functional differences with the human enzymes. Detailed kinetic analyses indicate that PfGluPho functions on the basis of a rapid equilibrium random Bi Bi mechanism, where the binding of the second substrate depends on the first substrate. We furthermore show that PfGluPho is inhibited by S-glutathionylation. The availability of recombinant PfGluPho and the major differences to hG6PD (human G6PD) facilitate studies on PfGluPho as an excellent drug target candidate in the search for new antimalarial drugs.
    Biochemical Journal 03/2011; 436(3):641-50. · 4.65 Impact Factor
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    ABSTRACT: The first step in the kynurenine pathway of tryptophan catabolism is the cleavage of the 2,3-double bond of the indole ring of tryptophan. In mammals, this reaction is performed independently by indoleamine 2,3-dioxygenase-1 (IDO1), tryptophan 2,3-dioxygenase (TDO) and the recently discovered indoleamine 2,3-dioxygenase-2 (IDO2). Here we describe characteristics of a purified recombinant mouse IDO2 enzyme, including its pH stability, thermal stability and structural features. An improved assay system for future studies of recombinant/isolated IDO2 has been developed using cytochrome b (5) as an electron donor. This, the first description of the interaction between IDO2 and cytochrome b (5), provides further evidence of the presence of a physiological electron carrier necessary for activity of enzymes in the "IDO family". Using this assay, the kinetic activity and substrate range of IDO2 were shown to be different to those of IDO1. 1-Methyl-D-tryptophan, a current lead IDO inhibitor used in clinical trials, was a poor inhibitor of both IDO1 and IDO2 activity. This suggests that its immunosuppressive effect may be independent of pharmacological inhibition of IDO enzymes, in the mouse at least. The different biochemical characteristics of the mouse IDO proteins suggest that they have evolved to have distinct biological roles.
    Amino Acids 02/2010; 39(2):565-78. · 3.91 Impact Factor
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    ABSTRACT: The malarial parasite Plasmodium falciparum possesses a functional thioredoxin and glutathione system comprising the dithiol-containing redox proteins thioredoxin (Trx) and glutaredoxin (Grx), as well as plasmoredoxin (Plrx), which is exclusively found in Plasmodium species. All three proteins belong to the thioredoxin superfamily and share a conserved Cys-X-X-Cys motif at the active site. Only a few of their target proteins, which are likely to be involved in redox reactions, are currently known. The aim of the present study was to extend our knowledge of the Trx-, Grx-, and Plrx-interactome in Plasmodium. Based on the reaction mechanism, we generated active site mutants of Trx and Grx lacking the resolving cysteine residue. These mutants were bound to affinity columns to trap target proteins from P. falciparum cell extracts after formation of intermolecular disulfide bonds. Covalently linked proteins were eluted with dithiothreitol and analyzed by mass spectrometry. For Trx and Grx, we were able to isolate 17 putatively redox-regulated proteins each. Furthermore, the approach was successfully established for Plrx, leading to the identification of 21 potential target proteins. In addition to confirming known interaction partners, we captured potential target proteins involved in various processes including protein biosynthesis, energy metabolism, and signal transduction. The identification of three enzymes involved in S-adenosylmethionine (SAM) metabolism furthermore suggests that redox control is required to balance the metabolic fluxes of SAM between methyl-group transfer reactions and polyamine synthesis. To substantiate our data, the binding of the redoxins to S-adenosyl-L-homocysteine hydrolase and ornithine aminotransferase (OAT) were verified using BIAcore surface plasmon resonance. In enzymatic assays, Trx was furthermore shown to enhance the activity of OAT. Our approach led to the discovery of several putatively redox-regulated proteins, thereby contributing to our understanding of the redox interactome in malarial parasites.
    PLoS Pathogens 05/2009; 5(4):e1000383. · 8.14 Impact Factor
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    ABSTRACT: Adenylate kinases (AK; ATP+AMP<-->2 ADP; E.C. 2.7.4.3.) are enzymes essentially involved in energy metabolism and macromolecular biosynthesis. As we reported previously, the malarial parasite Plasmodium falciparum possesses one genuine AK and one GTP-AMP phosphotransferase. Analysis of the P. falciparum genome suggested the presence of one additional adenylate kinase, which we designated AK2. Recombinantly produced AK2 was found to be a monomeric protein of 33 kDa showing a specific activity of 10 U/mg with ATP and AMP as a substrate pair and to interact with the AK-specific inhibitor P(1),P(5)-(diadenosine-5')-pentaphosphate (IC(50)=200 nM). At its N-terminus AK2 carries a predicted myristoylation sequence. This sequence is only present in AK2 of P. falciparum causing the severe tropical malaria and not in other malarial parasites. We heterologously coexpressed AK2 and P. falciparum N-myristoyltransferase (NMT) in the presence of myristate in Escherichia coli. As demonstrated by protein purification and mass spectrometry, AK2 is indeed myristoylated under catalysis of the parasites' transferase. The modification significantly enhances the stability of the kinase. Furthermore, AK2 and NMT were shown to interact strongly with each other forming a heterodimeric protein in vitro. To our knowledge this is the first direct evidence that P. falciparum NMT myristoylates an intact malarial protein.
    Molecular and Biochemical Parasitology 10/2008; 163(2):77-84. · 2.73 Impact Factor
  • Kathrin Buchholz, Boniface Mwongela Mailu, R. Heiner Schirmer, Katja Becker
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    ABSTRACT: The protozoon Plasmodium falciparum is the causative agent of tropical malaria which causes up to three million human deaths and up to 500 million episodes of clinical illness throughout the world annually. Children in African countries bear the largest part of this burden. Due to the rapid development of resistance to clinically used drugs like chloroquine and mefloquine and the increasing risk of resistance to artemisinins, novel effective and affordable antimalarial agents are urgently required. The progress made over the last years in the fields of genomics, proteomics, and clinical medicine coupled with improved facilities as well as technical progress in structural biology and high throughput screening methods are essential to support these drug development approaches. Furthermore concerted programs supported by governments, industry and academia contribute significantly to the progress in the field of antimalarial chemotherapy. Among the most interesting antimalarial target proteins currently studied are proteases, like plasmepsins, falcipains and falcilysin, but also protein kinases, glycolytic enzymes and enzymes involved in lipid metabolism and DNA replication. In addition, redox active proteins like glutathione reductase, thioredoxin reductase and glutathione S-transferase have become increasingly interesting. In this article we summarize the major current structure-based antimalarial drug development approaches. We briefly review the presently available three-dimensional structures of Plasmodium proteins together with their potential as drug targets. In parallel, we give an overview over inhibitors that have been developed on the basis of these known parasite protein structures or related structures of proteins from other organisms.
    Frontiers in Drug Design & Discovery: Structure-Based Drug Design in the 21st Century. 02/2007; 3(1):225-255.
  • Mailu BM, Katja Becker
    Internetmagazin der Bundesregierung zur Entwicklungspolitik. 01/2007; e.velop 52.
  • Boniface Mwongela Mailu
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    ABSTRACT: Plasmodium parasites are developing unacceptable levels of resistance to one drug after another and many insecticides are no longer useful against mosquitoes transmitting the disease. Years of vaccine research have produced few hopeful candidates and although scientists are redoubling the search, an effective vaccine is at best years away. Therefore there is need for identification of new drug targets and alternative antimalarial regimes. In response to this dire situation the study aimed at evaluating the pentose phosphate pathway of the malaria parasite P. falciparum in particular the bifunctional enzyme glucose-6-phosphate dehydrogenase- -phosphogluconolactonase, understanding the kynurenine pathway of tryptophan metabolism in particular the enzymes indoleamine 2,3-dioxygenase (1 and 2) and unravelling more knowledge about the thioredoxin system networks in search for a new potential drug target and new drug alternatives. The first two steps of the pentose phosphate pathway in Plasmodium falciparum are catalysed by the enzyme glucose 6-phosphate dehydrogenase-6-phosphogluconolactonase (PfGluPho) which is a unique bifunctional enzyme exclusively found in the genus Plasmodium. In spite of the importance of the role this enzyme plays in the parasite’s pentose phosphate pathway as well as in overcoming oxidative stress, the characteristics of PfGluPho are still a mystery. For the first time PfGluPho has been successfully cloned, heterologously overexpressed and purified to homogeneity. The recombinant enzyme was found to be a hexamer which exhibits lower Km values that favour substrate turnover by the parasite enzyme when compared to the human homologue. The steady state kinetics of PfGluPho’s glucose-6-phosphate dehydrogenase (PfGluPho’s G6PD) demonstrates that the enzyme follows an ordered sequential mechanism with NADP+ being the leading substrate. Three novel inhibitors of PfGluPho’s G6PD which are active at the lower micromolar range were identified and found to be non-competitive with respect to glucose-6-phosphate and NADP+. The study offers the first clear documentation of the cloning, heterologous overexpression, biochemical as well as kinetic characterisation, crystallisation and the first novel inhibitors of PfGluPho. For 30 years, the established dogma regarding tryptophan catabolism was that the first step of the kynurenine pathway, the cleavage of the 2,3–double bond of the indole ring of tryptophan was performed by two enzymes, indoleamine 2,3-dioxygenase-1 (IDO-1) and tryptophan 2,3-dioxygenase (TDO). Recently, indoleamine 2,3-dioxygenase-2 (IDO-2) a third enzyme capable of performing this reaction has been discovered. Reported here is a study of the kinetic activity, pH stability, oligomeric structure as well as secondary structural features of recombinant mouse IDO-2 in direct comparison with mouse IDO-1. A screen for new more potent inhibitors of IDO-1 which lack the indole core and avoid the liability arising from the use of indole derivatives which have been reported to be neuroactive gave rise to compound 55D11 (Ki 0.05 ìM) which is more potent than the already existing IDO inhibitors. A structure activity study was done using various derivatives of compound 55D11 to determine the elements that could be modified to increase potency. The study clearly demonstrates that IDO-1 and IDO-2 differ significantly in terms of their affinity for substrates as well as structure. The malarial parasite Plasmodium falciparum possesses a functional glutathione and thioredoxin system comprising the redox-active proteins thioredoxin (Trx), glutaredoxin (Grx), and plasmoredoxin (Plrx) which all belong to the thioredoxin superfamily and share the active site motif Cys-X-X-Cys. A better understanding of the role of these members of the thioredoxin superfamily in P. falciparum as well as other systems could be achieved if more was known about their target proteins. Using thioredoxin affinity chromatography prepared by immobilising mutants of the redoxins lacking the resolving cysteine at the active site on CNBr- activated sepharose, target proteins of P. falciparum cell extract were trapped. The covalently linked proteins were eluted with dithiothreitol and analyzed by matrix assisted laser desorption ionization time of flight (MALDI-TOF). Twenty one potential targets were identified for plasmoredoxin. Besides confirming known interacting proteins, potential target candidates involved in processes such as; protein biosynthesis, energy metabolism and signal transduction were identified. Further confirmations of the interaction of plasmoredoxin and the target proteins were done using BIAcore surface plasmon resonance experiments. Der Malaria-Parasit Plasmodium entwickelt bemerkenswert hohe Resistenzen gegenüber einem Medikament nach dem anderen. Außerdem verlieren viele Insektizide, die gegen die Überträger-Moskitos eingesetzt werden, an Wirkung. Jahrelange Forschung an Impfstoffen gegen Malaria hat bisher nur wenige hoffnungsvolle Kandidaten erbracht und obwohl Wissenschaftler Ihre Bemühungen verstärken, ist eine effektive Impfung bestenfalls immer noch Jahre entfernt. Deshalb sind dringend neue Zielmoleküle für die Medikamentenentwicklung zu identifizieren, die zu alternativen Behandlungsmethoden führen können. Diese Situation vor Augen, waren es Ziele dieser Arbeit, i) das bifunktionelle Enzym Glukose-6-Phosphatdehydrogenase-6-Phosphogluconolactonase aus dem Pentosephosphatweg des Parasiten als potentielles Wirkungsziel zu bestätigen, ii) den Kynurenin-Stoffwechselweg, insbesondere die Enzyme Indolamin 2,3-Dioxygenase (1 und 2) der Maus als Modell näher zu charakterisieren und iii) mehr über das Redox-Netzwerk des Malariaparasiten zu erfahren, um neue mögliche Zielmoleküle aufzuzeigen. Die ersten zwei Schritte des Pentosephosphatweges werden in Plasmodium falciparum durch das Enzym Glukose-6-Phosphatdehydrogenase-6-Phosphogluconolactonase (PfGluPho) katalysiert. Dieses ist ein einzigartiges bifunktionelles Enzym, das bisher nur in Plasmodien gefunden wurde. Obwohl diesem Enzym im Pentosephosphatweg des Parasiten und damit auch in der Bekämpfung oxidativen Stresses eine enorme Bedeutung zukommt, ist das Enzym aus P. falciparum nicht besonders gut charakterisiert, da es bisher nicht kloniert werden konnte. Zum ersten Mal konnte PfGluPho jetzt kloniert und überexprimiert werden und das Genprodukt konnte bis zur Reinheit gebracht werden. Es wurde gezeigt, dass das rekombinante Enzym als Hexamer vorliegt, welches niedrigere Km-Werte im Vergleich zu seinen humanen Orthologen aufweist, die einen bevorzugten Substratumsatz durch das parasitäre Enzym aufzeigen. Kinetische Untersuchungen zeigen, dass der Glukose-6-Phosphatdehydrogenase (G6PD)-Teil von PfGluPho einem geordneten Mechanismus folgt, bei dem NADP+ das erste Substrat ist. Drei neue Inhibitoren, die den G6PD-Teil des Enzym im unteren mikromolaren Konzentrationsbereich hemmen, konnten gefunden werden und zeigten sich gegenüber Glukose-6-Phosphat und NADP+ als nicht-kompetitiv. Somit zeigt diese Arbeit die Klonierung, heterologe Expression, biochemische und kinetische Charakterisierung von PfGluPho auf, sowie darüberhinaus die Kristallisation und erste, neue Inhibitoren. Seit 30 Jahren ist es ein etabliertes Dogma, dass im Tryptophan-Katabolismus der erste Schritt des Kynurenin-Stoffwechselweges die 2,3-Doppelbindung des Indolringes des Tryptophans durch zwei Enzyme gespalten werden kann: einerseits durch Indolamin 2,3- Dioxigenase (IDO), anderseits durch Tryptophan 2,3-Dioxigenase (TDO). Kürzlich konnte mit IDO-2 ein drittes Enzym in der Maus und im Menschen entdeckt werden, das in der Lage ist, diese Reaktion zu vollziehen. In dieser Arbeit sind Daten zur Kinetik, pH-Stabilität, zu oligomeren Strukturen, sowie Besonderheiten der Sekundärstruktur von rekombinanter IDO-2 x der Maus im direkten Vergleich mit rekombinanter IDO-1 der Maus erhoben worden. Die Suche nach neuen, effektiveren Inhibitoren für IDO-1, die den Indolkern nicht mehr besitzen und somit weniger neurotoxisch sein könnten, führte zu 55D11 (Ki 0,05 µM), einer Substanz aus einer öffentlichen Sammlung von Naturstoffen. Zahlreiche Derivate von 55D11 wurden untersucht, um Molekülreste zu zeigen, die verändert werden können und die Aktivität noch erhöhen. Die Untersuchungen zeigen, dass sich IDO-1 und IDO-2 eindeutig hinsichtlich ihrer Affinität zu Substraten, aber auch in ihrer Struktur unterscheiden. Der Malaria-Parasit Plasmodium falciparum besitzt ein funktionelles Glutathion-, sowie ein Thioredoxin-System, die unter anderem aus den redox-aktiven Proteinen Thioredoxin (Trx), Glutaredoxin (Grx) und Plasmoredoxin (Plrx) bestehen, die alle zur sogenannten Thioredoxin-Superfamilie gehören und das Motiv Cys-X-X-Cys im aktiven Zentrum besitzen. Wenn mehr Interaktionspartner dieser Redoxproteine bekannt wären, könnte die jeweilige Rolle der Redoxine in ihrem Netzwerk besser verstanden werden. Deshalb wurden Thioredoxin-Affinitätschromatographien an CNBr--aktivierter Sepharose durchgeführt. Hierzu wurden immobilisierte Mutanten der Redoxine, denen das sogenannte "resolving cysteine" aus ihrem aktiven Zentrum fehlt, mit Zellextrakt aus P. falciparum versetzt. Kovalent an die Fängerproteine (Redoxine) gebundene Reaktanden wurden mit Dithiothreitol eluiert und mittels (Maldi-ToF) analysiert. Einundzwanzig potentielle Zielproteine wurden für Plasmoredoxin als mögliche Interaktionspartner identifiziert. Neben bekannten Interaktionspartnern waren darunter auch Kandidaten, die eine mögliche Redoxregulierung der Proteinbiosynthese, des Energiemetabolismus sowie der Signaltransduktion in Plasmodium vermuten lassen können. Weitere Untersuchungen, um diese Wechselwirkungen zu bestätigen, wurden mit Plasmoredoxin und einigen seiner potentiellen Interaktionspartner mithilfe von BIACORE Oberflächen-Resonanzexperimenten durchgeführt.