Jean-Michel Jault

Publications (33)117.26 Total impact

• Article: Characterization of YvcC (BmrA), a multidrug ABC transporter constitutively expressed in Bacillus subtilis.

  Emmanuelle Steinfels, Cédric Orelle, Jean-Raphaël Fantino, Olivier Dalmas, Jean-Louis Rigaud, François Denizot, Attilio Di Pietro, Jean-Michel Jault
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  ABSTRACT: The involvement of transporters in multidrug resistance of bacteria is an increasingly challenging problem, and most of the pumps identified so far use the protonmotive gradient as the energy source. A new member of the ATP-binding cassette (ABC) family, known in Bacillus subtilis as YvcC and homologous to each half of mammalian P-glycoprotein and to LmrA of Lactococcus lactis, has been studied here. The yvcC gene was constitutively expressed in B. subtilis throughout its growth, and a knockout mutant showed a lower rate of ethidium efflux than the wild-type strain. Overexpression of yvcC in Escherichia coli allowed the preparation of highly enriched inverted-membrane vesicles that exhibited high transport activities of three fluorescent drugs, namely, Hoechst 33342, doxorubicin, and 7-aminoactinomycin D. After solubilization with n-dodecyl beta-D-maltoside, the hexahistidine-tagged YvcC was purified by a one-step affinity chromatography, and its ability to bind many P-glycoprotein effectors was evidenced by fluorescence spectroscopy experiments. Collectively, these results showed that YvcC is a multidrug ABC transporter functionally active in wild-type B. subtilis, and YvcC was therefore renamed BmrA for Bacillus multidrug resistance ATP. Besides, reconstitution of YvcC into liposomes led to the highest, vanadate-sensitive, ATPase activity reported so far for an ABC transporter. Interestingly, such a high ATP hydrolysis proceeds with a positive cooperativity mechanism, a property only found so far with ABC importers.
  Biochemistry 07/2004; 43(23):7491-502. · 3.42 Impact Factor
• Article: Two FHA domains on an ABC transporter, Rv1747, mediate its phosphorylation by PknF, a Ser/Thr protein kinase from Mycobacterium tuberculosis.

  Virginie Molle, Didier Soulat, Jean-Michel Jault, Christophe Grangeasse, Alain J Cozzone, Jean-François Prost
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  ABSTRACT: Bacterial genomics have revealed the widespread occurrence of eukaryotic-like protein kinases in prokaryotes, but little is known about their regulation, endogenous substrates, and physiological role. The present study concerns one of these enzymes, the serine/threonine protein kinase PknF from Mycobacterium tuberculosis. It is shown that, in addition to its autokinase activity, PknF is able to phosphorylate Rv1747, a newly described ABC transporter. This reaction appears to involve two FHA domains of Rv1747. It is suggested that recruitment and phosphorylation of Rv1747 depend on the interaction between its two non-redundant FHA domains and the autophosphorylated form of PknF.
  FEMS Microbiology Letters 06/2004; 234(2):215-23. · 2.04 Impact Factor
• Article: The conserved glutamate residue adjacent to the Walker-B motif is the catalytic base for ATP hydrolysis in the ATP-binding cassette transporter BmrA.

  Cédric Orelle, Olivier Dalmas, Philippe Gros, Attilio Di Pietro, Jean-Michel Jault
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  ABSTRACT: ATP-binding cassette (ABC) proteins constitute one of the widest families in all organisms, whose P-glycoprotein involved in resistance of cancer cells to chemotherapy is an archetype member. Although three-dimensional structures of several nucleotide-binding domains of ABC proteins are now available, the catalytic mechanism triggering the functioning of these proteins still remains elusive. In particular, it has been postulated that ATP hydrolysis proceeds via an acid-base mechanism catalyzed by the Glu residue adjacent to the Walker-B motif (Geourjon, C., Orelle, C., Steinfels, E., Blanchet, C., Deléage, G., Di Pietro, A., and Jault, J. M. (2001) Trends Biochem. Sci. 26, 539-544), but the involvement of such residue as the catalytic base in ABC transporters was recently questioned (Sauna, Z. E., Muller, M., Peng, X. H., and Ambudkar, S. V. (2002) Biochemistry, 41, 13989-14000). The equivalent glutamate residue (Glu504) of a half-ABC transporter involved in multidrug resistance in Bacillus subtilis, BmrA (formerly known as YvcC), was therefore mutated to Asp, Ala, Gln, Ser, and Cys residues. All these mutants were fully devoid of ATPase activity, yet they showed a high level of vanadate-independent trapping of 8-N3-alpha-32P-labeled nucleotide(s), following preincubation with 8-N3-[alpha-32P]ATP. However, and in contrast to the wild-type enzyme, the use of 8-N3-[gamma-32P]ATP unequivocally showed that all the mutants trapped exclusively the triphosphate form of the analogue, suggesting that they were not able to perform even a single hydrolytic turnover. These results demonstrate that Glu504 is the catalytic base for ATP hydrolysis in BmrA, and it is proposed that equivalent glutamate residues in other ABC transporters play the same role.
  Journal of Biological Chemistry 12/2003; 278(47):47002-8. · 4.77 Impact Factor
• Article: Regulation and mutational analysis of the HPr kinase/phosphorylase from Bacillus subtilis.

  Frédérique Pompeo, Yohann Granet, Jean-Pierre Lavergne, Christophe Grangeasse, Sylvie Nessler, Jean-Michel Jault, Anne Galinier
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  ABSTRACT: In most Gram-positive bacteria, catabolite repression is mediated by a bifunctional enzyme, the HPr kinase/phosphorylase (HprK/P). It has recently been shown that HprK/P could catalyze the phosphorylation of the protein HPr by using pyrophosphate (PP(i)) as a phosphate donor instead of ATP. Here we showed that, as for ATP, PP(i) binds to the enzyme with strong positive cooperativity. However, in contrast to ATP, PP(i) binding does not modify the fluorescence properties of the unique Trp residue of Bacillus subtilis HprK/P. In addition, to understand how two conserved motifs, namely, the P-loop and the specific signature of this family, participate in the three enzymatic activities of HprK/Ps (ATP-kinase, PP(i)-kinase, and phosphorylase), several site-directed mutants were generated. Whereas the three activities are mediated by the P-loop which is directly involved in the binding of ATP, PP(i), or Pi, the signature motif seems to be involved preferentially in the dephosphorylation reaction. On the basis of these results, we propose a model in which the binding of the allosteric activator FBP induces a conformational change of a central loop located above the active site of HprK/P, thereby allowing the ATP binding. However, this conformational change is not required for the binding of PP(i).
  Biochemistry 07/2003; 42(22):6762-71. · 3.42 Impact Factor
• Article: Highly efficient over-production in E. coli of YvcC, a multidrug-like ATP-binding cassette transporter from Bacillus subtilis.

  Emmanuelle Steinfels, Cédric Orelle, Olivier Dalmas, François Penin, Bruno Miroux, Attilio Di Pietro, Jean-Michel Jault
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  ABSTRACT: ATP-binding cassette (ABC) transporters have often been refractory to over-expression. Using the C41(DE3) E. coli as a host strain, membrane vesicles highly enriched (>50%) in YvcC, a previously uncharacterized ABC transporter from Bacillus subtilis homologous to P-glycoprotein multidrug transporters, were obtained. The functionality of YvcC was assessed by its high vanadate-sensitive ATPase activity and its ability to transport a fluorescent drug, the Hoechst 33342.
  Biochimica et Biophysica Acta 09/2002; 1565(1):1-5. · 4.66 Impact Factor
• Article: Insights into the functioning of Bacillus subtilis HPr kinase/phosphatase: affinity for its protein substrates and role of cations and phosphate.

  Jean-Pierre Lavergne, Jean-Michel Jault, Anne Galinier
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  ABSTRACT: In Bacillus subtilis, carbon catabolite repression is mediated by the HPr kinase/phosphatase (HprK/P) which catalyzes both an ATP-dependent phosphorylation and a dephosphorylation on Ser-46 of either HPr (histidine-containing protein) or Crh (catabolite repression HPr). By using a surface plasmon resonance approach, it was shown here that the presence of magnesium is a prerequisite for the interaction of HprK/P with either HPr or Crh. HprK/P binds both protein substrates with a similar affinity (K(D) of about 40 nM), and addition of nucleotides increases by about 10-fold its affinity for each substrate. In addition, the specificity and the concentration of the cation required for the binding of protein substrates are different from that exhibited by the cation-binding site involved in the nucleotide binding, suggesting the presence of two cation-binding sites on HprK/P. The effects of phosphate on enzymatic activities of HprK/P were also investigated. Phosphate was able to unmask the phosphatase activity, especially in the presence of ATP or both ATP and fructose 1,6-bisphosphate whereas it was shown to inhibit the kinase activity of HprK/P. An apparent competition between phosphate and a fluorescent analogue of nucleotide led to the suggestion that phosphate mediates its effect by binding directly to the ATP-binding site of the enzyme.
  Biochemistry 06/2002; 41(20):6218-25. · 3.42 Impact Factor
• Article: A new family of phosphotransferases with a P-loop motif.

  Anne Galinier, Jean-Pierre Lavergne, Christophe Geourjon, Sonia Fieulaine, Sylvie Nessler, Jean-Michel Jault
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  ABSTRACT: In most Gram-positive bacteria, catabolite repression is mediated by a bifunctional enzyme, the histidine-containing protein kinase/phosphatase (HprK/P). Based either on its primary sequence or on its recently solved three-dimensional structure, no straightforward homology with other known proteins was found. However, we showed here that HprK/P exhibits a restricted homology with an unrelated phosphotransferase, the phosphoenolpyruvate carboxykinase. This includes notably two consecutive Asp residues from the phosphoenolpyruvate carboxykinase active site, whose equivalent residues were mutated in Bacillus subtilis HprK/P. Characterization of the corresponding mutants emphasizes the crucial role of these Asp residues in the HprK/P functioning. Furthermore, superimposition of HprK/P and phosphoenolpyruvate carboxykinase active sites supports the view that both enzymes bear significant resemblance in their overall mechanism of functioning showing that these two enzymes constitute a new family of phosphotransferases.
  Journal of Biological Chemistry 04/2002; 277(13):11362-7. · 4.77 Impact Factor
• Article: Three-dimensional structure by cryo-electron microscopy of YvcC, an homodimeric ATP-binding cassette transporter from Bacillus subtilis.

  Mohamed Chami, Emmanuelle Steinfels, Cédric Orelle, Jean-Michel Jault, Attilio Di Pietro, Jean-Louis Rigaud, Sergio Marco
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  ABSTRACT: YvcC, a multidrug transporter from Bacillus subtilis, is a member of the ATP-binding cassette superfamily, highly homologous to each half of human multidrug-resistance P-glycoprotein and to several other bacterial half-ABC transporters. Here, the purified recombinant histidine-tagged YvcC has been reconstituted into a lipid bilayer. Controlled and partial detergent removal from YvcC-lipid micelles allowed the production of particularly interesting lipid-detergent-YvcC ring-shaped particles, about 40 nm in diameter, well suited for single particle analysis by cryo-electron microscopy. Furthermore, binding of these histidine-tagged ring-shaped particles to lipid layers functionalized with a Ni(2+)-chelating head group generated a preferential perpendicular orientation, eliminating the missing cone in the final three-dimensional reconstruction. From such analysis, a computed volume has been determined to 2.5 nm resolution giving a detailed insight into the structural organization of this half-ABC transporter within a membrane. The repetitive unit in the ring-shaped particles is consistent with a homodimeric organization of YvcC. Each subunit was composed of three domains: a 5 nm height transmembrane region, a stalk of about 4 nm in height and 2 nm in diameter, and a cytoplasmic lobe of about 5-6 nm in diameter. The latest domain, which fitted with the reported X-ray structure of HisP, was identified as the nucleotide-binding domain (NBD). The 3D reconstruction of the YvcC homodimer well compared with the very recent X-ray crystallographic data on the MsbA homodimer from Escherichia coli, supporting the existence of a central open chamber between the two subunits constituting the homodimer. In addition, the 3D reconstruction of YvcC embedded in a membrane revealed an asymmetric organization of the two NBDs sites within the homodimer, as well as a dimeric interaction between two homodimers.
  Journal of Molecular Biology 03/2002; 315(5):1075-85. · 4.00 Impact Factor
• Article: The isocitrate dehydrogenase kinase/phosphatase from Escherichia coli is highly sensitive to in‐vitro oxidative conditions

  Christelle Oudot, Michel Jaquinod, Jean-Claude Cortay, Alain J. Cozzone, Jean-Michel Jault
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  ABSTRACT: Isocitrate dehydrogenase kinase/phosphatase (IDHK/P) is a homodimeric enzyme which controls the oxidative metabolism of Escherichia coli, and exibits a high intrinsic ATPase activity. When subjected to electrophoresis under nonreducing conditions, the purified enzyme migrates partially as a dimer. The proportion of the dimer over the monomer is greatly increased by treatment with cupric 1,10 phenanthrolinate or 5,5′-dithio-bis(2-nitrobenzoic acid), and fully reversed by dithiothreitol, indicating that covalent dimerization is produced by a disulfide bond. To identify the residue(s) involved in this intermolecular disulfide-bond, each of the eight cysteines of the enzyme was individually mutated into a serine. It was found that, under nonreducing conditions, the electrophoretic patterns of all corresponding mutants are identical to that of the wild-type, except for the Cys67→Ser which migrates exclusively as a monomer and for the Cys108→Ser which migrates preferentially as a dimer. Furthermore, in contrast to the wild-type enzyme and all the other mutants, the Cys67→Ser mutant still migrates as a monomer after treatment with cupric 1,10 phenanthrolinate. This result indicates that the intermolecular disulfide bond involves only Cys67 in each IDHK/P wild-type monomer. This was further supported by mass spectrum analysis of the tryptic peptides derived from either the cupric 1,10 phenanthrolinate-treated wild-type enzyme or the native Cys108→Ser mutant, which show that they both contain a Cys67–Cys67 disulfide bond. Moreover, both the cupric 1,10 phenanthrolinate-treated wild-type enzyme and the native Cys108→Ser mutant contain another disulfide bond between Cys356 and Cys480. Previous results have shown that this additional Cys356–Cys480 disulfide bond is intramolecular [Oudot, C., Jault, J.-M., Jaquinod, M., Negre, D., Prost, J.-F., Cozzone, A.J. & Cortay, J.-C. (1998) Eur. J. Biochem. 258, 579–585].
  European Journal of Biochemistry. 12/2001; 262(1):224 - 229.
• Article: The “Catalytic” Triad of Isocitrate Dehydrogenase Kinase/Phosphatase from E. coli and Its Relationship with That Found in Eukaryotic Protein Kinases

  Christelle Oudot, Jean-Claude Cortay, Christophe Blanchet, David C. Laporte, Attilio Di Pietro, Alain J. Cozzone, Jean-Michel Jault
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  ABSTRACT: The isocitrate dehydrogenase kinase/phosphatase (IDHK/P) of E. coli is a bifunctional enzyme responsible for the reversible phosphorylation of isocitrate dehydrogenase (IDH) on a seryl residue. As such, it belongs to the serine/threonine protein kinase family. However, only a very limited homology with the well-characterized eukaryotic members of that family was identified so far in its primary structure. In this report, a new region of amino acids including three putative residues involved in the kinase activity of IDHK/P was identified by sequence comparison with eukaryotic protein kinases. In IDHK/P, these residues are Asp-371, Asn-377, and Asp-403. Their counterpart eukaryotic residues have been shown to be involved in either catalysis (former residue) or magnesium binding (the two latter residues). Site-directed mutagenesis was performed on these three IDHK/P residues, and also on the Glu-439 residue equivalent to that of the Ala-Pro-Glu motif found in the eukaryotic protein kinases. Mutations of Asp-371 into either Ala, Glu, or Gln residues drastically lowered the yield and the quality of the purification. Nevertheless, the recovered mutant enzymes were barely able to phosphorylate IDH either in vitro or after expression in an aceK - mutant strain. In contrast, mutation of either Asn-377, Asp-403, or Glu-439 into an Ala residue altered neither the yield of purification nor the maximal phosphorylating capacity of the enzyme. However, when IDH was phosphorylated in the presence of increasing concentrations of magnesium ions, the two former mutants displayed a much lower affinity for this cation, with a Km value of 0.6 or 0.8 mM, respectively, as compared to 0.1 mM for the wild-type enzyme. On the other hand, the Glu439Ala mutant has an affinity for magnesium essentially unaffected. Therefore, and in contrast to the current opinion, our results suggest that the catalytic mechanism of IDHK/P exhibits some similarities with that found in the eukaryotic members of the protein kinase family.
  02/2001;
• Article: Inactivation of isocitrate dehydrogenase kinase/phosphatase by 5′‐[p‐(fluorosulfonyl)benzoyl]adenosine is not due to the labeling of the invariant lysine residue found in the protein kinase family

  Christelle Oudot, Jean-Michel Jault, Michel Jaquinod, Didier Negre, Jean-François Prost, Alain J. Cozzone, Jean-Claude Cortay
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  ABSTRACT: The ATPase activity of Escherichia coli isocitrate dehydrogenase kinase/phosphatase was rapidly lost after prior incubation with the ATP analogue 5′-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA). This inactivation was prevented by the presence of either 5 mM ATP or 5 mM ADP plus Mg2+, while it could be fully reversed by subsequent addition of dithiothreitol, thereby indicating the involvement of cysteine residue(s) in this process. About 2 mol [3H]FSBA/mol IDHK/P were bound during the time course of the inactivation. However, this binding was not significantly modified by either prior incubation with ATP or subsequent addition of dithiothreitol. This suggested that FSBA-mediated inactivation of isocitrate dehydrogenase kinase/phosphatase occurred via the formation of a disulfide bond. Accordingly, mass spectral analysis revealed that on addition of FSBA, a disulfide bond was formed between residues Cys356 and Cys523. The mutation Cys356Ser renders the enzyme insensitive to FSBA treatment indicating that Cys356 is the primary target for this analogue. However, the Cys523Ser mutant was still inactivated by FSBA and mass spectral analysis showed that this was due to the formation of a new disulfide bond between Cys356 and Cys480.
  European Journal of Biochemistry. 11/1998; 258(2):579 - 585.
• Article: Preparation of highly phosphorylating mitochondria from the yeastSchizosaccharomyces pombe

  Jean-Michel Jault, Jane Comte, Danièle C. Gautheron, Attilio Pietro
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  ABSTRACT: Schizosaccharomyces pombe yeast cells grown on either fermentable or respiratory media were efficiently converted to stable spheroplasts by the -(1 3)-glucanase Novozym 234 in the presence of 1.2M sorbitol. Lysis of spheroplasts by gentle homogenization in dilute sorbitol resulted in the preparation of mitochondria with a structure similar to that observed within the starting yeast cells. The isolated mitochondria exhibited high oxidation rates with various respiratory substrates, NADH being the most efficient. The mitochondria appeared well coupled since the second State 4 rate observed after ADP consumption was identical to the initial one. The State 3 rate in the presence of ADP was completely inhibited by low oligomycin concentrations, similarly to the concomitant ATP synthesis of 900 nmol/min mg protein. These NADH oxidation and dependent ATP-synthesis activities are much higher than those previously described for mitochondria isolated fromSchizosaccharomyces pombe, and similar to the highest values reported forSaccharomyces cerevisiae.
  Journal of Bioenergetics 07/1994; 26(4):447-456. · 2.81 Impact Factor
• Article: Purification from a yeast mutant of mitochondrial F1 with modified β-subunit: High affinity for nucleotides and high negative cooperativity of ATPase activity

  Pierre Falson, Attilio Di Pietro, Jean-Michel Jault, Danièle C. Gautheron, Marc Boutry
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  ABSTRACT: Mitochondrial F1 containing genetically modified β-subunit was purified for the first time from a mutant of the yeast Schizosaccharomyces pombe. Precipitation by poly(ethylene glycol) allowed us to obtain a very stable and pure enzyme from either mutant or wild-type strain. In the presence of EDTA, purified F1 retained high amounts of endogenous nucleotides: 4.6 mol/mol and 3.7 mol/mol for mutant and wild-type F1, respectively. The additional nucleotide in mutant F1 was ATP; it was lost in the presence of Mg2+, which led to a totalof 3.4 mol of nucleotides/mol whereas wild-type F1 retained all its nucleotides. Mutant F1 bound more exogenous ADP than wild-type F1 and the same total nucleotide amount was reached with both enzymes. Kinetics of ATPase activity revealed a much higher negative cooperativity for mutant than for wild-type F1. Bicarbonate abolished this negative cooperativity, but higher concentrations were required for mutant F1. The mutant enzyme was more sensitive than the wild-type one to azide inhibition and ADP competitive inhibition; this indicated stronger interactions between nucleotide and F1 in the mutant enzyme. The latter also showed increased sensitivity to N,N′ dicyclohexylcarbodiimide irreversible inhibition.
  Biochimica et Biophysica Acta (BBA) - Bioenergetics.