Biomedical subjects

P A Michels

Publications and source records attributed to P A Michels.

At least 19 recordsLinked to original sources

PubMed2001

NMR spectroscopic analysis of the first two steps of the pentose-phosphate pathway elucidates the role of 6-phosphogluconolactonase.

The pentose-phosphate pathway provides reductive power and nucleotide precursors to the cell through oxidative and nonoxidative branches, respectively. 6-Phosphogluconolactonase is the second enzyme of the oxidative branch and catalyzes the hydrolysis of 6-phosphogluconolactones, the products of glucose 6-phosphate oxidation by glucose-6-phosphate dehydrogenase. The role of 6-phosphogluconolactonase was still questionable, because 6-phosphogluconolactones were believed to undergo rapid spontaneous hydrolysis. In this work, nuclear magnetic resonance spectroscopy was used to characterize the chemical scheme and kinetic features of the oxidative branch. We show that 6-phosphogluconolactones have in fact a nonnegligible lifetime and are highly electrophilic compounds. The delta form (1-5) of the lactone is the only product of glucose 6-phosphate oxidation. Subsequently, it leads to the gamma form (1-4) by intramolecular rearrangement. However, only the delta form undergoes spontaneous hydrolysis, the gamma form being a "dead end" of this branch. The delta form is the only substrate for 6-phosphogluconolactonase. Therefore, 6-phosphogluconolactonase activity accelerates hydrolysis of the delta form, thus preventing its conversion into the gamma form. Furthermore, 6-phosphogluconolactonase guards against the accumulation of delta-6-phosphogluconolactone, which may be toxic through its reaction with endogenous cellular nucleophiles. Finally, the difference between activity of human, Trypanosoma brucei, and Plasmodium falciparum 6-phosphogluconolactonases is reported and discussed.

Carboxylic Ester Hydrolases↗

PubMed2001

Enzymes of carbohydrate metabolism as potential drug targets.

The potential for chemotherapeutic exploitation of carbohydrate metabolism in the Trypanosomatidae is reviewed. This review is based largely on discussions held at a meeting of the COST B9 Action, entitled 'Bioenergetics of Protozoan Parasites'. The major questions posed were: which enzymes are the best to target; what further information is required to allow their use for rational drug development; what compounds would constitute the best inhibitors and which of the enzymes of the pentose-phosphate pathway are present inside the glycosomes, as well? Only partial answers could be obtained in many cases, but the interactive discussion between the multidisciplinary group of participants, comprising chemists, biochemists and molecular biologists, provided thought-provoking ideas and will help direct future research.

Animals↗

PubMed2001

An unexpected extended conformation for the third TPR motif of the peroxin PEX5 from Trypanosoma brucei.

A number of helix-rich protein motifs are involved in a variety of critical protein-protein interactions in living cells. One of these is the tetratrico peptide repeat (TPR) motif that is involved, amongst others, in cell cycle regulation, chaperone function and post-translation modifications. So far, these helix-rich TPR motifs have always been observed to be a compact unit of two helices interacting with each other in antiparallel fashion. Here, we describe the structure of the first three TPR-motifs of the peroxin PEX5 from Trypanosoma brucei, the causative agent of sleeping sickness. Peroxins are proteins involved in peroxisome, glycosome and glyoxysome biogenesis. PEX5 is the receptor of the proteins targeted to these organelles by the "peroxisomal targeting signal-1", a C-terminal tripeptide called PTS-1. The first two of the three TPR-motifs of T. brucei PEX5 appear to adopt the canonical antiparallel helix hairpin structure. In contrast, the third TPR motif of PEX5 has a dramatically different conformation in our crystals: the two helices that were supposed to form a hairpin are folded into one single 44 A long continuous helix. Such a conformation has never been observed before for a TPR motif. This raises interesting questions including the potential functional importance of a "jack-knife" conformational change in TPR motifs.

Amino Acid Motifs↗

PubMed2001

The expression and intracellular distribution of phosphoglycerate kinase isoenzymes in Trypanosoma cruzi.

In this paper, we report the subcellular distribution of phosphoglycerate kinase (PGK) in epimastigotes of Trypanosoma cruzi. Approximately 80% of the PGK activity was found in the cytosol, 20% in the glycosomes. Western blot analysis suggested that two isoenzymes of 56 and 48 kDa, respectively, are responsible for the glycosomal PGK activity, whereas the cytosolic activity should be attributed to a single PGK of 48 kDa. In analogy to the situation previously reported for PGK in Trypanosoma brucei, these isoenzymes were called PGKA, C and B, respectively. However, in T. cruzi, PGKA seems not to be a minor enzyme like its counterpart in T. brucei. Whereas PGKC behaved as a soluble glycosomal matrix protein, PGKA appeared to be present at the inner surface of the organelle's membrane. After alkaline carbonate treatment, the enzyme remained associated with the particulate fraction of the organelles. Upon solubilization of glycosomes with Triton X-114, PGKA was recovered from the detergent phase, indicating its (partial) hydrophobic character and therefore, a possible hydrophobic interaction with the membrane. The PGKA gene was cloned and sequenced, but the predicted amino-acid sequence did not reveal an obvious clue as to the mechanism by which the enzyme is attached to the glycosomal membrane.

Amino Acid Sequence↗

PubMed2001

Glycolysis as a target for the design of new anti-trypanosome drugs.

Glycolysis is perceived as a promising target for new drugs against parasitic trypanosomatid protozoa because this pathway plays an essential role in their ATP supply. Trypanosomatid glycolysis is unique in that it is compartmentalized, and many of its enzymes display unique structural and kinetic features. Structure- and catalytic mechanism-based approaches are applied to design compounds that inhibit the glycolytic enzymes of the parasites without affecting the corresponding proteins of the human host. For some trypanosomatid enzymes, potent and selective inhibitors have already been developed that affect only the growth of cultured trypanosomatids, and not mammalian cells.

Animals↗

PubMed2000

A family of highly conserved glycosomal 2-hydroxyacid dehydrogenases from Phytomonas sp.

Phytomonas sp. contains two malate dehydrogenase isoforms, a mitochondrial isoenzyme with a high specificity for oxaloacetate and a glycosomal isozyme that acts on a broad range of substrates (Uttaro, A. D., and Opperdoes, F.R. (1997) Mol. Biochem. Parasitol. 89, 51-59). Here, we show that the low specificity of the latter isoenzyme is the result of a number of recent gene duplications that gave rise to a family of glycosomal 2-hydroxyacid dehydrogenase genes. Two of these genes were cloned, sequenced, and overexpressed in Escherichia coli. Although both gene products have 322 amino acids, share 90.4% identical residues, and have a similar hydrophobicity profile and net charge, their kinetic properties were strikingly different. One isoform behaved as a real malate dehydrogenase with a high specificity for oxaloacetate, whereas the other showed no activity with oxaloacetate but was able to reduce other oxoacids, such as phenyl pyruvate, 2-oxoisocaproate, 2-oxovalerate, 2-oxobutyrate, 2-oxo-4-methiolbutyrate, and pyruvate.

Alcohol Oxidoreductases↗

PubMed2000

Molecular characterization of the first two enzymes of the pentose-phosphate pathway of Trypanosoma brucei. Glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase.

Trypanosomatids are parasitic protists that have part of their glycolytic pathway sequestered inside peroxisome-like organelles: the glycosomes. So far, at least one enzyme of the pentose-phosphate pathway has been found to be associated partially with glycosomes. Here, we describe how two genes from Trypanosoma brucei, coding for the first two enzymes of the pentose-phosphate pathway, i.e. glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase, were identified by in silico screening of trypanosome genome project data bases. These genes were cloned and sequenced. Analysis of the lactonase sequence revealed that it contained a C-terminal peroxisome targeting signal in agreement with its subcellular localization in the bloodstream form trypanosome (15% glycosomal and 85% cytosolic). However, the dehydrogenase sequence did not reveal any targeting signal, despite its localization inside glycosomes. The corresponding enzymes have been overexpressed in Escherichia coli and purified, and their biochemical characteristics have been determined.

Amino Acid Sequence↗

PubMed2000

Structures of type 2 peroxisomal targeting signals in two trypanosomatid aldolases.

Trypanosomatids, unicellular organisms responsible for several global diseases, contain unique organelles called glycosomes in which the first seven glycolytic enzymes are sequestered. We report the crystal structures of glycosomal fructose-1,6-bisphosphate aldolase from two major tropical pathogens, Trypanosoma brucei and Leishmania mexicana, the causative agents of African sleeping sickness and one form of leishmaniasis, respectively. Unlike mammalian aldolases, the T. brucei and L. mexicana aldolases contain nonameric N-terminal type 2 peroxisomal targeting signals (PTS2s) to direct their import into the glycosome. In both tetrameric trypanosomatid aldolases, the PTS2s from two different subunits form two closely intertwined structures. These "PTS2 dimers", which have very similar conformations in the two aldolase structures, are the first reported conformations of a glycosomal or peroxisomal PTS2, and provide opportunities for the design of trypanocidal compounds.

Amino Acid Sequence↗

PubMed2000

A potential target enzyme for trypanocidal drugs revealed by the crystal structure of NAD-dependent glycerol-3-phosphate dehydrogenase from Leishmania mexicana.

BACKGROUND: NAD-dependent glycerol-3-phosphate dehydrogenase (GPDH) catalyzes the interconversion of dihydroxyacetone phosphate and L-glycerol-3-phosphate. Although the enzyme has been characterized and cloned from a number of sources, until now no three-dimensional structure has been determined for this enzyme. Although the utility of this enzyme as a drug target against Leishmania mexicana is yet to be established, the critical role played by GPDH in the long slender bloodstream form of the related kinetoplastid Trypanosoma brucei makes it a viable drug target against sleeping sickness. RESULTS: The 1.75 A crystal structure of apo GPDH from L. mexicana was determined by multiwavelength anomalous diffraction (MAD) techniques, and used to solve the 2.8 A holo structure in complex with NADH. Each 39 kDa subunit of the dimeric enzyme contains a 189-residue N-terminal NAD-binding domain and a 156-residue C-terminal substrate-binding domain. Significant parts of both domains share structural similarity with plant acetohydroxyacid isomeroreductase. The discovery of extra, fatty-acid like, density buried inside the C-terminal domain indicates a possible post-translational modification with an associated biological function. CONCLUSIONS: The crystal structure of GPDH from L. mexicana is the first structure of this enzyme from any source and, in view of the sequence identity of 63%, serves as a valid model for the T. brucei enzyme. The differences between the human and trypanosomal enzymes are extensive, with only 29% sequence identity between the parasite and host enzyme, and support the feasibility of exploiting the NADH-binding site to develop selective inhibitors against trypanosomal GPDH. The structure also offers a plausible explanation for the observed inhibition of the T. brucei enzyme by melarsen oxide, the active form of the trypanocidal drugs melarsoprol and cymelarsan.

Animals↗

PubMed2000

Compartmentation protects trypanosomes from the dangerous design of glycolysis.

Unlike in other organisms, in trypanosomes and other Kinetoplastida the larger part of glycolysis takes place in a specialized organelle, called the glycosome. At present it is impossible to remove the glycosome without changing much of the rest of the cell. It would seem impossible, therefore, to assess the metabolic consequences of this compartmentation. Therefore, we here develop a computer experimentation approach, which we call computational cell biology. A validated molecular kinetic computer replica was built of glycolysis in the parasite Trypanosoma brucei. Removing the glycosome membrane in that replica had little effect on the steady-state flux, which argues against the prevalent speculation that glycosomes serve to increase flux by concentrating the enzymes. Removal of the membrane did cause (i) the sugar phosphates to rise to unphysiologically high levels, which must have pathological effects, and (ii) a failure to recover from glucose deprivation. We explain these effects on the basis of the biochemical organization of the glycosome. We conclude (i) that the glycosome protects trypanosomes from the negative side effects of the "turbo" structure of glycolysis and (ii) that computer experimentation based on solid molecular data is a powerful tool to address questions that are not, or not yet, accessible to experimentation.

Adenosine Triphosphate↗

PubMed2000

Metabolic control analysis of glycolysis in trypanosomes as an approach to improve selectivity and effectiveness of drugs.

Glycolysis is the only ATP-generating process in bloodstream form trypanosomes and is therefore a promising drug target. Inhibitors which decrease significantly the glycolytic flux will kill the parasites. Both computer simulation and experimental studies of glycolysis in bloodstream form Trypanosoma brucei indicated that the control of the glycolytic flux is shared by several steps in the pathway. The results of these analyses provide quantitative information about the prospects of decreasing the flux by inhibition of any individual enzyme. The plasma membrane glucose transporter appears the most promising target from this perspective, followed by aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and glycerol-3-phosphate dehydrogenase. Non-competitive or irreversible inhibitors would be most effective, but it is argued that potent competitive inhibitors can be suitable, provided that the concentration of the competing substrate cannot increase unrestrictedly. Such is the case for inhibitors that compete with coenzymes or with blood glucose.

Animals↗

PubMed2000

Comparative study of Leishmania mexicana and Trypanosoma brucei NAD-dependent glycerol-3-phosphate dehydrogenase.

The NAD-dependent glycerol-3-phosphate dehydrogenases (G3PDH, EC 1.1.1.8) of Trypanosoma brucei and Leishmania mexicana are thought to have different roles in carbohydrate metabolism. Here the physicochemical and kinetic properties of natural G3PDH from T. brucei with the recombinant homologue of L. mexicana which share 63% positional identity are compared. Despite their supposed different functions in energy metabolism of the parasites the two G3PDHs have remarkably similar properties, including pH optima and K(m) value for dihydroxyacetone phosphate (DHAP) and NADH in the formation of glycerol 3-phosphate (G3P) and for NAD+ and G3P in the reverse reaction. Both enzymes are subject inhibition by dihydroxyacetone phosphate at concentrations above 0.2 mM and are inhibited by the trypanocidal drugs suramin and melarsen oxide at sub-micromolar concentrations.

Animals↗

PubMed2000

Metabolic aspects of glycosomes in trypanosomatidae - new data and views.

The energy metabolism of Trypanosomatidae has been the subject of many reviews during the past decade. In recent years, however, new data have led to a more complete picture of trypanosomatid metabolism and a reappraisal of the role of some characteristic organelles in the energy supply of these parasites. For years, the glycosome was thought to be a peroxisome-like organelle that had evolved to allow the parasites to carry out glycolysis at a high rate using a relatively small amount of enzyme. However, the results of recent studies of trypanosomatid glycolysis and the detection of various other pathways and enzymes in the organelle necessitate a modification of this view. Here, Paul Michels, Véronique Hannaert and Frédéric Bringaud review the new data and discuss the possible implications for our view on the role of the glycosome.

Animals↗

PubMed2000

Leishmania pyruvate kinase: the crystal structure reveals the structural basis of its unique regulatory properties.

Glycolysis occupies a central role in cellular metabolism, and is of particular importance for the catabolic production of ATP in protozoan parasites such as Leishmania and Trypanosoma. In these organisms pyruvate kinase plays a key regulatory role, and is unique in responding to fructose 2,6-bisphosphate as allosteric activator. The determination of the crystal structure of the first eukaryotic pyruvate kinase in the T-state (the inactive or 'tense' conformation of allosteric enzymes) is described. A comparison of the effector sites of the Leishmania and yeast enzymes reveals the structural basis for the different effector specificity. Two loops, comprising residues 443-453 and 480-489, adopt very different conformations in the two enzymes, and Lys-453 and His-480 that are a feature of trypanosomatid enzymes provide probable ligands for the 2-phospho group of the effector molecule. These and other differences offer an opportunity for the design of drugs that would exploit regulatory differences between parasite and host.

Amino Acid Sequence↗

PubMed2000

Trypanosoma brucei contains a 2,3-bisphosphoglycerate independent phosphoglycerate mutase.

Assays of phosphoglycerate mutase (PGAM) activity in lysates of bloodstream form Trypanosoma brucei appeared not to require exogenous 2,3-bisphosphoglycerate, thus suggesting that this protist contains an enzyme belonging to the class of cofactor-independent PGAMs. A gene encoding a polypeptide with motifs characteristic for this class of enzymes was cloned. The predicted T. brucei PGAM polypeptide contains 549 amino acids, with Mr 60 557 and pI 5.5. Comparison with 15 cofactor-independent PGAM sequences available in databases showed that the amino-acid sequence of the trypanosome enzyme has 59-62% identity with plant PGAMs and 29-35% with eubacterial enzymes. A low 28% identity was observed with the only available invertebrate sequence. The trypanosome enzyme has been expressed in Escherichia coli, purified to homogeneity and subjected to preliminary kinetic analysis. Previous studies have shown that cofactor-dependent and -independent PGAMs are not homologous. It has been inferred that the cofactor-independent PGAMs are in fact homologous to a family of metalloenzymes containing alkaline phosphatases and sulphatases. Prediction of the secondary structure of T. brucei PGAM and threading the sequence into the known crystal structure of E. coli alkaline phosphatase (AP) confirmed this homology, despite the very low sequence identity. Generally, a good match between predicted (PGAM) and actual (AP) secondary structure elements was observed. In contrast to trypanosomes, glycolysis in all vertebrates involves a cofactor-dependent PGAM. The presence of distinct nonhomologous PGAMs in the parasite and its human host offers great potential for the design of selective inhibitors which could form leads for new trypanocidal drugs.

2,3-Diphosphoglycerate↗

PubMed2000

Glycerol kinase of Trypanosoma brucei. Cloning, molecular characterization and mutagenesis.

Trypanosoma brucei contains two tandemly arranged genes for glycerol kinase. The downstream gene was analysed in detail. It contains an ORF for a polypeptide of 512 amino acids. The polypeptide has a calculated molecular mass of 56 363 Da and a pI of 8.6. Comparison of the T. brucei glycerol kinase amino-acid sequence with the glycerol kinase sequences available in databases revealed positional identities of 39.0-50.4%. The T. brucei glycerol kinase gene was overexpressed in Escherichia coli cells and the recombinant protein obtained was purified and characterized biochemically. Its kinetic properties with regard to both the forward and reverse reaction were measured. The values corresponded to those determined previously for the natural glycerol kinase purified from the parasite, and confirmed that the apparent Km values of the trypanosome enzyme for its substrates are relatively high compared with those of other glycerol kinases. Alignment of the amino-acid sequences of T. brucei glycerol kinase and other eukaryotic and prokaryotic glycerol kinases, as well as inspection of the available three-dimensional structure of E. coli glycerol kinase showed that most residues of the magnesium-, glycerol- and ADP-binding sites are well conserved in T. brucei glycerol kinase. However, a number of remarkable substitutions was identified, which could be responsible for the low affinity for the substrates. Most striking is amino-acid Ala137 in T. brucei glycerol kinase; in all other organisms a serine is present at the corresponding position. We mutated Ala137 of T. brucei glycerol kinase into a serine and this mutant glycerol kinase was over-expressed and purified. The affinity of the mutant enzyme for its substrates glycerol and glycerol 3-phosphate appeared to be 3. 1-fold to 3.6-fold higher than in the wild-type enzyme. Part of the glycerol kinase gene comprising this residue 137 was amplified in eight different kinetoplastid species and sequenced. Interestingly, an alanine occurs not only in T. brucei, but also in other trypanosomatids which can convert glucose into equimolar amounts of glycerol and pyruvate: T. gambiense, T. equiperdum and T. evansi. In trypanosomatids with no or only a limited capacity to produce glycerol, a hydroxy group-containing residue is found as in all other organisms: T. vivax and T. congolense possess a serine while Phytomonas sp., Leishmania brasiliensis and L. mexicana have a threonine.

Amino Acid Sequence↗

PubMed1999

Molecular characterisation of Trypanosoma brucei alkyl dihydroxyacetone-phosphate synthase.

Alkyl dihydroxyacetone-phosphate synthase is the second enzyme of the ether-lipid biosynthetic pathway which is responsible for the introduction of the ether linkage between a fatty alcohol and a glycerol present in a subclass of phospholipids, the plasmalogens and possibly in glycolipid membrane anchors. In this study the gene coding for alkyl dihydroxyacetone-phosphate synthase was isolated from Trypanosoma brucei. Southern blot analysis of total genomic DNA suggested the presence of a single copy gene. The analysis, together with sequencing of different cDNA clones showed that the two alleles of the gene differ in only one nucleotide. The gene encodes a protein of 612 amino acids with a calculated molecular mass of 68,891, not counting the initiator methionine. It carries a type-1 peroxisomal targeting signal (a C-terminal tripeptide--AHL) and a calculated overall positive charge of +10. The gene was expressed in a bacterial system and the corresponding protein carrying a His-tag was purified. The recombinant alkyl dihydroxyacetone-phosphate synthase and the enzyme isolated directly from the glycosomes of bloodstream-form trypanosomes have comparable kinetics. The Km for hexadecanol was 42 microM, while approximately 100 microM of palmitoyl dihydroxyacetone phosphate (DHAP) was necessary for optimal activity. Sodium chloride inhibited both the His-tagged protein and the enzyme isolated from the glycosomes of bloodstream-form and insect stage T. brucei.

Alkyl and Aryl Transferases↗

PubMed1999

Cloning and analysis of the PTS-1 receptor in Trypanosoma brucei.

Kinetoplastid organisms, such as the protozoan parasite Trypanosoma brucei, compartmentalise several important metabolic pathways in organelles called glycosomes. Glycosomes are related to peroxisomes of yeast and mammalian cells. A subset of glycosomal matrix proteins is routed to the organelles via the peroxisome-targeting signal type 1 (PTS-1). The PEX5 gene homologue has been cloned from T. brucei coding for a protein of the translocation machinery, the PTS-1 receptor. The gene codes for a polypeptide of 654 amino acids with a calculated molecular mass of 70 kDa. Like its homologue in other organisms T. brucei PTS-1 receptor protein (TbPEX5) is a member of the tetratricopeptide repeat (TPR) protein family and contains several copies of the pentapeptide W-X-X-X-F/Y. Northern and Western blot analysis showed that the protein is expressed at different stages of the life cycle of the parasite. The protein has been overproduced in Escherichia coli and purified using immobilized metal affinity chromatography. The purified protein specifically interacts in vitro with glycosomal phosphoglycerate kinase-C (PGK-C) of T. brucei, a PTS-1 containing protein. The equilibrium dissociation constant (Kd) of PGK-C for purified TbPEX5 is 40 nM. Using biochemical and cytochemical techniques a predominantly cytosolic localization was found for TbPEX5. This is consistent with the idea of receptor cycling between the glycosomes and the cytosol.

Amino Acid Sequence↗