Pierre Le Maréchal

Transmembrane modulator-dependent bacterial tyrosine kinase activates UDP-glucose dehydrogenases

Protein‐tyrosine kinases regulating bacterial exopolysaccharide synthesis autophosphorylate on tyrosines located in a conserved C‐terminal region. So far no other substrates have been identified for these kinases. Here we demonstrate that Bacillus subtilis YwqD not only autophosphorylates at Tyr‐228, but that it also phosphorylates the two UDP‐glucose dehydrogenases (UDP‐glucose DHs) YwqF and TuaD at a tyrosine residue. However, phosphorylation of YwqF and TuaD occurs only in the presence of the transmembrane protein YwqC. The presumed intracellular C‐terminal part of YwqC (last 50 amino acids) seems to interact with the tyrosine‐kinase and to allow YwqD‐catalysed phosphorylation of the two UDP‐glucose DHs, which are key enzymes for the synthesis of acidic polysaccharides. However, only when phosphorylated by YwqD do the two enzymes exhibit detectable UDP‐glucose DH activity. Dephosphorylation of P‐Tyr‐YwqF and P‐Tyr‐TuaD by the P‐Tyr‐protein phosphatase YwqE switched off their UDP‐glucose DH activity. YwqE, which is encoded by the fourth gene of the B.subtilis ywqCDEF operon, also dephosphorylates P‐Tyr‐YwqD.

The thioredoxin-independent isoform of chloroplastic glyceraldehyde-3-phosphate dehydrogenase is selectively regulated by glutathionylation

In animal cells, many proteins have been shown to undergo glutathionylation under conditions of oxidative stress. By contrast, very little is known about this post‐translational modification in plants. In the present work, we showed, using mass spectrometry, that the recombinant chloroplast A4‐glyceraldehyde‐3‐phosphate dehydrogenase (A4‐GAPDH) from Arabidopsis thaliana is glutathionylated with either oxidized glutathione or reduced glutathione and H2O2. The formation of a mixed disulfide between glutathione and A4‐GAPDH resulted in the inhibition of enzyme activity. A4‐GAPDH was also inhibited by oxidants such as H2O2. However, the effect of glutathionylation was reversed by reductants, whereas oxidation resulted in irreversible enzyme inactivation. On the other hand, the major isoform of photosynthetic GAPDH of higher plants (i.e. the AnBn‐GAPDH isozyme in either A2B2 or A8B8 conformation) was sensitive to oxidants but did not seem to undergo glutathionylation significantly. GAPDH catalysis is based on Cys149 forming a covalent intermediate with the substrate 1,3‐bisphosphoglycerate. In the presence of 1,3‐bisphosphoglycerate, A4‐GAPDH was fully protected from either oxidation or glutathionylation. Site‐directed mutagenesis of Cys153, the only cysteine located in close proximity to the GAPDH active‐site Cys149, did not affect enzyme inhibition by glutathionylation or oxidation. Catalytic Cys149 is thus suggested to be the target of both glutathionylation and thiol oxidation. Glutathionylation could be an important mechanism of regulation and protection of chloroplast A4‐GAPDH from irreversible oxidation under stress.

An Internal Cysteine Is Involved in the Thioredoxin-dependent Activation of Sorghum Leaf NADP-malate Dehydrogenase

The chloroplastic NADP-malate dehydrogenase is activated by thiol/disulfide interchange with reduced thioredoxins. Previous experiments showed that four cysteines located in specific N- and carboxyl-terminal extensions were implicated in this process, leading to a model where no internal cysteine was involved in activation. In the present study, the role of the conserved four internal cysteines was investigated. Surprisingly, the mutation of cysteine 207 into alanine yielded a protein with accelerated activation time course, whereas the mutations of the three other internal cysteines into alanines yielded proteins with unchanged activation kinetics. These results suggested that cysteine 207 might be linked in a disulfide bridge with one of the four external cysteines, most probably with one of the two amino-terminal cysteines whose mutation similarly accelerates the activation rate. To investigate this possibility, mutant malate dehydrogenases (MDHs) where a single amino-terminal cysteine was mutated in combination with the mutation of both carboxyl-terminal cysteines were produced and purified. The C29S/C365A/C377A mutant MDH still needed activation by reduced thioredoxin, while the C24S/C365A/C377A mutant MDH exhibited a thioredoxin-insensitive spontaneous activity, leading to the hypothesis that a Cys24-Cys207 disulfide bridge might be formed during the activation process. Indeed, an NADP-MDH where the cysteines 29, 207, 365, and 377 are mutated yielded a permanently active enzyme very similar to the previously created permanently active C24S/C29S/C365A/C377A mutant. A two-step activation model involving a thioredoxin-mediated disulfide isomerization at the amino terminus is proposed.

Functional Properties of Sarcoplasmic Reticulum Ca2+-ATPase after Proteolytic Cleavage at Leu119-Lys120, Close to the A-domain

By measuring the phosphorylation levels of individual proteolytic fragments of SERCA1a separated by electrophoresis after their phosphorylation, we were able to study the catalytic properties of a p95C-p14N complex arising from SERCA1a cleavage by proteinase K between Leu119 and Lys120, in the loop linking the A-domain with the second transmembrane segment. ATP hydrolysis by the complex was very strongly inhibited, although ATP-dependent phosphorylation and the conversion of the ADP-sensitive E1P form to E2P still occurred at appreciable rates. However, the rate of subsequent dephosphorylation of E2P was inhibited to a dramatic extent, and this was also the case for the rate of “backdoor” formation of E2P from E2 and Pi. E2P formation from E2 at equilibrium nevertheless indicated little change in the apparent affinity for Pi or Mg2+, while binding of orthovanadate was weaker. The p95C-p14N complex also had a slightly reduced affinity for Ca2+ and exhibited a reduced rate for its Ca2+-dependent transition from E2 to Ca2E1. Thus, disruption of the N-terminal link of the A-domain with the transmembrane region seems to shift the conformational equilibria of Ca2+-ATPase from the E1/E1P toward the E2/E2P states and to increase the activation energy for dephosphorylation of Ca2+-ATPase, reviving the old idea of the A-domain being a phosphatase domain as part of the transduction machinery.

The phosphorylation of Sorghum leaf phosphoenolpyruyate carboxylase is a Ca++-calmodulin dependent process

Regulation of the in vitro phosphorylation process of the photosynthetic form (G form) of Sorghum leaf Phosphoenolpyruvate carboxylase (PEPC: EC 4.1.1.31) was studied. Results established that: 1) PEPC was efficiently phosphorylated on seryl residues in crude leaf extract 2) Pyruvate, orthophosphate dikinase (EC 2.7.9.1.) which has been supposed to interfere with the process, was found not to be significantly phosphorylated in our experimental conditions 3) KF, as well as both Ca++ and Mg++ ions increased the radioactive signal detected 4) addition of EDTA or EGTA nullified it and Ca++ alone was found to reverse the inhibitory effect exerted by both chelators 5) addition of anti-Calmodulin antibodies to the medium also abolished the PEPC phosphorylation. Present data demonstrated that the post-translational modification of the C4-plant photosynthetic PEPC is a Ca++/Calmodulin dependent process.

Compared properties of phosphoenolpyruvate carboxylase from dark- and light-adapted Sorghum leaves: use of a rapid purification technique by immunochromatography

Abstract Two C4-type phosphoenolpyruvate carboxylase (EC 4.1.1.31, PEPc) forms of Sorghum leaves, differing by their phosphorylation state (the phosphorylated form being predominant during the day), have been purified from dark- and light-adapted leaves by using an immunoaffinity chromatography column. The phosphorylation state of these purified PEPc forms was assessed by incubation with either the endogenous PEPc protein-serine kinase, or the catalytic subunit of the cAMP-dependent protein kinase and alkaline phosphatase on the dark and light enzyme, respectively. HPLC gel filtration studies showed that both forms are tetrameric at concentrations used throughout the work. Comparison of the kinetic properties at pH 7.0, 7.3 and 8.0, indicated a cooperativity for phosphoenolpyruvate (PEP) at all pHs considered, with a Hill number of 2 for both forms and higher Vm(2- to 4-fold) and Km for the light form. Malate inhibition mainly affected the Vm of the dark form, whereas it was an allosteric inhibitor for the light enzyme. The results established that phosphorylation is the molecular basis which mediates the functional properties of the enzyme. In a medium mimicking an illuminated mesophyll cytosol (2.5 mM PEP, 5 mM glucose-6-P, 20 mM malate and 10 mM MgCl2 (pH 7.3), the activity of the enzyme from light-adapted leaves was twice that of the dark one. This increased activity due to phosphorylation may be of importance for the regulation of C4 photosynthesis.

S-palmitoylation and s-oleoylation of rabbit and pig sarcolipin.

Background: Many functions are subjected to regulation by protein-protein interactions. An example is the SERCA1a-SLN8 complex. Results: Rabbit and pig SLN have two types of fatty acid anchors (palmitic and oleic acid) attached to an intramembranous cysteine residue. Conclusion: A role and evolutionary significance of these S-acylations are suggested. Significance: First demonstration of SLN S-acylation and of the intramembranous S-oleoylation of a membrane protein. Sarcolipin (SLN) is a regulatory peptide present in sarcoplasmic reticulum (SR) from skeletal muscle of animals. We find that native rabbit SLN is modified by a fatty acid anchor on Cys-9 with a palmitic acid in about 60% and, surprisingly, an oleic acid in the remaining 40%. SLN used for co-crystallization with SERCA1a (Winther, A. M., Bublitz, M., Karlsen, J. L., Moller, J. V., Hansen, J. B., Nissen, P., and Buch-Pedersen, M. J. (2013) Nature 495, 265–2691; Ref. 1) is also palmitoylated/oleoylated, but is not visible in crystal structures, probably due to disorder. Treatment with 1 m hydroxylamine for 1 h removes the fatty acids from a majority of the SLN pool. This treatment did not modify the SERCA1a affinity for Ca2+ but increased the Ca2+-dependent ATPase activity of SR membranes indicating that the S-acylation of SLN or of other proteins is required for this effect on SERCA1a. Pig SLN is also fully palmitoylated/oleoylated on its Cys-9 residue, but in a reverse ratio of about 40/60. An alignment of 67 SLN sequences from the protein databases shows that 19 of them contain a cysteine and the rest a phenylalanine at position 9. Based on a cladogram, we postulate that the mutation from phenylalanine to cysteine in some species is the result of an evolutionary convergence. We suggest that, besides phosphorylation, S-acylation/deacylation also regulates SLN activity.

Comparative Proteomic Analysis of Streptomyces lividans Wild-Type and ppk Mutant Strains Reveals the Importance of Storage Lipids for Antibiotic Biosynthesis

ABSTRACT Streptomyces lividans TK24 is a strain that naturally produces antibiotics at low levels, but dramatic overproduction of antibiotics occurs upon interruption of the ppk gene. However, the role of the Ppk enzyme in relation to the regulation of antibiotic biosynthesis remains poorly understood. In order to gain a better understanding of the phenotype of the ppk mutant, the proteomes of the wild-type (wt) and ppk mutant strains, grown for 96 h on R2YE medium limited in phosphate, were analyzed. Intracellular proteins were separated on two-dimensional (2D) gels, spots were quantified, and those showing a 3-fold variation or more were identified by mass spectrometry. The expression of 12 proteins increased and that of 29 decreased in the ppk mutant strain. Our results suggested that storage lipid degradation rather than hexose catabolism was taking place in the mutant. In order to validate this hypothesis, the triacylglycerol contents of the wt and ppk mutant strains of S. lividans as well as that of Streptomyces coelicolor M145, a strain that produces antibiotics at high levels and is closely related to S. lividans, were assessed using electron microscopy and thin-layer chromatography. These studies highlighted the large difference in triacylglycerol contents of the three strains and confirmed the hypothetical link between storage lipid metabolism and antibiotic biosynthesis in Streptomyces.

Vitamin K‐dependent carboxylation: inhibition by a peptide containing 4‐methylene glutamic acid

Vitamin K Carboxylation 4‐Methylene glutamic acid γ‐Carboxyglutamic acid

The Role of Active Site Arginines of Sorghum NADP-Malate Dehydrogenase in Thioredoxin-dependent Activation and Activity

The activation of sorghum NADP-malate dehydrogenase is initiated by thiol/disulfide interchanges with reduced thioredoxin followed by the release of the C-terminal autoinhibitory extension and a structural modification shaping the active site into a high efficiency and high affinity for oxaloacetate conformation. In the present study, the role of the active site arginines in the activation and catalysis was investigated by site-directed mutagenesis and arginyl-specific chemical derivatization using butanedione. Sequence and mass spectrometry analysis were used to identify the chemically modified groups. Taken together, our data reveal the involvement of Arg-134 and Arg-204 in oxaloacetate coordination, suggest an indirect role for Arg-140 in substrate binding and catalysis, and clearly confirm that Arg-87 is implicated in cofactor binding. In contrast with NAD-malate dehydrogenase, no lactate dehydrogenase activity could be promoted by the R134Q mutation. The decreased susceptibility of the activation of the R204K mutant to NADP and its increased sensitivity to the histidine-specific reagent diethylpyrocarbonate indicated that Arg-204 is involved in the locking of the active site. These results are discussed in relation with the recently published NADP-MDH three-dimensional structures and the previously established three-dimensional structures of NAD-malate dehydrogenase and lactate dehydrogenase.