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Guy Branlant

Publications and source records attributed to Guy Branlant.

At least 19 recordsLinked to original sources
PubMed2006

Invariant Thr244 is essential for the efficient acylation step of the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans.

One of the most striking features of several X-ray structures of CoA-independent ALDHs (aldehyde dehydrogenases) in complex with NAD(P) is the conformational flexibility of the NMN moiety. However, the fact that the rate of the acylation step is high in GAPN (non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase) from Streptococcus mutans implies an optimal positioning of the nicotinamide ring relative to the hemithioacetal intermediate within the ternary GAPN complex to allow an efficient and stereospecific hydride transfer. Substitutions of serine for invariant Thr244 and alanine for Lys178 result in a drastic decrease of the efficiency of hydride transfer which becomes rate-limiting. The crystal structure of the binary complex T244S GAPN-NADP shows that the absence of the beta-methyl group leads to a well-defined conformation of the NMN part, including the nicotinamide ring, clearly different from that depicted to be suitable for an efficient hydride transfer in the wild-type. The approximately 0.6-unit increase in pK(app) of the catalytic Cys302 observed in the ternary complex for both mutated GAPNs is likely to be due to a slight difference in positioning of the nicotinamide ring relative to Cys302 with respect to the wild-type ternary complex. Taken together, the data support a critical role of the Thr244 beta-methyl group, held in position through a hydrogen-bond interaction between the Thr244 beta-hydroxy group and the epsilon-amino group of Lys178, in permitting the nicotinamide ring to adopt a conformation suitable for an efficient hydride transfer during the acylation step for all the members of the CoA-independent ALDH family.

2,2'-Dipyridyl↗
PubMed2006

Functional and structural aspects of poplar cytosolic and plastidial type a methionine sulfoxide reductases.

The genome of Populus trichocarpa contains five methionine sulfoxide reductase A genes. Here, both cytosolic (cMsrA) and plastidial (pMsrA) poplar MsrAs were analyzed. The two recombinant enzymes are active in the reduction of methionine sulfoxide with either dithiothreitol or poplar thioredoxin as a reductant. In both enzymes, five cysteines, at positions 46, 81, 100, 196, and 202, are conserved. Biochemical and enzymatic analyses of the cysteine-mutated MsrAs support a catalytic mechanism involving three cysteines at positions 46, 196, and 202. Cys(46) is the catalytic cysteine, and the two C-terminal cysteines, Cys(196) and Cys(202), are implicated in the thioredoxin-dependent recycling mechanism. Inspection of the pMsrA x-ray three-dimensional structure, which has been determined in this study, strongly suggests that contrary to bacterial and Bos taurus MsrAs, which also contain three essential Cys, the last C-terminal Cys(202), but not Cys(196), is the first recycling cysteine that forms a disulfide bond with the catalytic Cys(46). Then Cys(202) forms a disulfide bond with the second recycling cysteine Cys(196) that is preferentially reduced by thioredoxin. In agreement with this assumption, Cys(202) is located closer to Cys(46) compared with Cys(196) and is included in a (202)CYG(204) signature specific for most plant MsrAs. The tyrosine residue corresponds to the one described to be involved in substrate binding in bacterial and B. taurus MsrAs. In these MsrAs, the tyrosine residue belongs to a similar signature as found in plant MsrAs but with the first C-terminal cysteine instead of the last C-terminal cysteine.

Cloning, Molecular↗
PubMed2006

Solution structure and backbone dynamics of the reduced form and an oxidized form of E. coli methionine sulfoxide reductase A (MsrA): structural insight of the MsrA catalytic cycle.

Methionine sulfoxide reductases (Msr) reduce methionine sulfoxide (MetSO)-containing proteins, back to methionine (Met). MsrAs are stereospecific for the S epimer whereas MsrBs reduce the R epimer of MetSO. Although structurally unrelated, the Msrs characterized so far display a similar catalytic mechanism with formation of a sulfenic intermediate on the catalytic cysteine and a concomitant release of Met, followed by formation of at least one intramolecular disulfide bond (between the catalytic and a recycling cysteine), which is then reduced by thioredoxin. In the case of the MsrA from Escherichia coli, two disulfide bonds are formed, i.e. first between the catalytic Cys51 and the recycling Cys198 and then between Cys198 and the second recycling Cys206. Three crystal structures including E. coli and Mycobacterium tuberculosis MsrAs, which, for the latter, possesses only the unique recycling Cys198, have been solved so far. In these structures, the distances between the cysteine residues involved in the catalytic mechanism are too large to allow formation of the intramolecular disulfide bonds. Here structural and dynamical NMR studies of the reduced wild-type and the oxidized (Cys51-Cys198) forms of C86S/C206S MsrA from E. coli have been carried out. The mapping of MetSO substrate-bound C51A MsrA has also been performed. The data support (1) a conformational switch occurring subsequently to sulfenic acid formation and/or Met release that would be a prerequisite to form the Cys51-Cys198 bond and, (2) a high mobility of the C-terminal part of the Cys51-Cys198 oxidized form that would favor formation of the second Cys198-Cys206 disulfide bond.

Binding Sites↗
PubMed2006

Characterization of the amino acids from Neisseria meningitidis MsrA involved in the chemical catalysis of the methionine sulfoxide reduction step.

Methionine sulfoxide reductases (Msrs) are ubiquitous enzymes that reduce protein-bound methionine sulfoxide back to Met in the presence of thioredoxin. In vivo, the role of the Msrs is described as essential in protecting cells against oxidative damages and as playing a role in infection of cells by pathogenic bacteria. There exist two structurally unrelated classes of Msrs, called MsrA and MsrB, specific for the S and the R epimer of the sulfoxide function of methionine sulfoxide, respectively. Both Msrs present a similar catalytic mechanism, which implies, as a first step, a reductase step that leads to the formation of a sulfenic acid on the catalytic cysteine and a concomitant release of a mole of Met. The reductase step has been previously shown to be efficient and not rate-limiting. In the present study, the amino acids involved in the catalysis of the reductase step of the Neisseria meningitidis MsrA have been characterized. The invariant Glu-94 and to a lesser extent Tyr-82 and Tyr-134 are shown to play a major role in the stabilization of the sulfurane transition state and indirectly in the decrease of the pK(app) of the catalytic Cys-51. A scenario of the reductase step is proposed in which the substrate binds to the active site with its sulfoxide function largely polarized via interactions with Glu-94, Tyr-82, and Tyr-134 and participates via the positive or partially positive charge borne by the sulfur of the sulfoxide in the stabilization of the catalytic Cys.

Cysteine↗
PubMed2006

Mechanistic characterization of the MSDH (methylmalonate semialdehyde dehydrogenase) from Bacillus subtilis.

Homotetrameric MSDH (methylmalonate semialdehyde dehydrogenase) from Bacillus subtilis catalyses the NAD-dependent oxidation of MMSA (methylmalonate semialdehyde) and MSA (malonate semialdehyde) into PPCoA (propionyl-CoA) and acetyl-CoA respectively via a two-step mechanism. In the present study, a detailed mechanistic characterization of the MSDH-catalysed reaction has been carried out. The results suggest that NAD binding elicits a structural imprinting of the apoenzyme, which explains the marked lag-phase observed in the activity assay. The enzyme also exhibits a half-of-the-sites reactivity, with two subunits being active per tetramer. This result correlates well with the presence of two populations of catalytic Cys302 in both the apo- and holo-enzymes. Binding of NAD causes a decrease in reactivity of the two Cys302 residues belonging to the two active subunits and a pKapp shift from approx. 8.8 to 8.0. A study of the rate of acylation as a function of pH revealed a decrease in the pKapp of the two active Cys302 residues to approx. 5.5. Taken to-gether, these results support a sequential Cys302 activation process with a pKapp shift from approx. 8.8 in the apo-form to 8.0 in the binary complex and finally to approx. 5.5 in the ternary complex. The rate-limiting step is associated with the b-decarboxylation process which occurs on the thioacylenzyme intermediate after NADH release and before transthioesterification. These data also indicate that bicarbonate, the formation of which is enzyme-catalysed, is the end-product of the reaction.

2,2'-Dipyridyl↗
PubMed2006

The first crystal structure of a thioacylenzyme intermediate in the ALDH family: new coenzyme conformation and relevance to catalysis.

Crystal structures of several members of the nonphosphorylating CoA-independent aldehyde dehydrogenase (ALDH) family have shown that the peculiar binding mode of the cofactor to the Rossmann fold results in a conformational flexibility for the nicotinamide moiety of the cofactor. This has been hypothesized to constitute an essential feature of the catalytic mechanism because the conformation of the cofactor required for the acylation step is not appropriate for the deacylation step. In the present study, the structure of a reaction intermediate of the E268A-glyceraldehyde 3-phosphate dehydrogenase (GAPN) from Streptococcus mutans, obtained by soaking the crystals of the enzyme/NADP complex with the natural substrate, is reported. The substrate is bound covalently in the four monomers and presents the geometric characteristics expected for a thioacylenzyme intermediate. Control experiments assessed that reduction of the coenzyme has occurred within the crystal. The structure reveals that reduction of the cofactor upon acylation leads to an extensive motion of the nicotinamide moiety with a flip of the reduced pyridinium ring away from the active site without significant changes of the protein structure. This event positions the reduced nicotinamide moiety in a pocket that likely constitutes the exit door for NADPH. Arguments are provided that the structure reported here constitutes a reasonable picture of the first thioacylenzyme intermediate characterized thus far in the ALDH family and that the position of the reduced nicotinamide moiety observed in GAPN is the one suitable for the deacylation step within all of the nonphosphorylating CoA-independent ALDH family.

Acylation↗
PubMed2006

The X-ray structure of the N-terminal domain of PILB from Neisseria meningitidis reveals a thioredoxin-fold.

The secreted form of the PilB protein was recently shown to be bound to the outer membrane of Neisseria gonorrhoeae and proposed to be involved in survival of the pathogen to the host's oxidative burst. PilB is composed of three domains. The central and the C-terminal domains display methionine sulfoxide reductase (Msr) A and B activities respectively, i.e. the ability to reduce specifically the S and the R enantiomers of the sulfoxide function of the methionine sulfoxides, which are easily formed upon oxidation of methionine residues. The N-terminal domain of PilB (Dom1(PILB)) of N.meningitidis, which possesses a CXXC motif, was recently shown to recycle the oxidized forms of the PilB Msr domains in vitro, as the Escherichia coli thioredoxin (Trx) 1 does. The X-ray structure of Dom1(PILB) of N.meningitidis determined here shows a Trx-fold, in agreement with the biochemical properties of Dom1(PILB). However, substantial structural differences with E.coli Trx1 exist. Dom1(PILB) displays more structural homologies with the periplasmic disulfide oxidoreductases involved in cytochrome maturation pathways in bacteria. The active site of the reduced form of Dom1(PILB) reveals a high level of stabilization of the N-terminal catalytic cysteine residue and a hydrophobic environment of the C-terminal recycling cysteine in the CXXC motif, consistent with the pK(app) values measured for Cys67 (<6) and Cys70 (9.3), respectively. Compared to cytochrome maturation disulfide oxidoreductases and to Trx1, one edge of the active site is covered by four additional residues (99)FLHE(102). The putative role of the resulting protuberance is discussed in relation to the disulfide reductase properties of Dom1(PILB).

Amino Acid Sequence↗
PubMed2005

Thermal destabilization of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans upon phosphate binding in the active site.

Catalysis by the NADP-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Streptococcus mutans, a member of the aldehyde dehydrogenase (ALDH) family, relies on a local conformational reorganization of the active site. This rearrangement is promoted by the binding of NADP and is strongly kinetically favored by the formation of the ternary complex enzyme.NADP.substrate. Adiabatic differential scanning calorimetry was used to investigate the effect of ligands on the irreversible thermal denaturation of GAPN. We showed that phosphate binds to GAPN, resulting in the formation of a GAPN.phosphate binary complex characterized by a strongly decreased thermal stability, with a difference of at least 15 degrees C between the maximum temperatures of the thermal transition peaks. The kinetics of phosphate association and dissociation are slow, allowing both free and GAPN.phosphate complexes to be observed by differential scanning calorimetry and to be separated by native polyacrylamide electrophoresis run in phosphate buffer. Analysis of a set of mutants of GAPN strongly suggests that phosphate is bound to the substrate C-3 subsite. In addition, the substrate analog glycerol-3-phosphate has similar effects as does phosphate on the thermal behavior of GAPN. Based on the current knowledge on the catalytic mechanism of GAPN and other ALDHs, we propose that ligand-induced thermal destabilization is a mechanism that provides to ALDHs the required flexibility for an efficient catalysis.

Binding Sites↗
PubMed2005

Misfolded forms of glyceraldehyde-3-phosphate dehydrogenase interact with GroEL and inhibit chaperonin-assisted folding of the wild-type enzyme.

We studied the interaction of chaperonin GroEL with different misfolded forms of tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH): (1) GAPDH from rabbit muscles with all SH-groups modified by 5,5'-dithiobis(2-nitrobenzoate); (2) O-R-type dimers of mutant GAPDH from Bacillus stearothermophilus with amino acid substitutions Y283V, D282G, and Y283V/W84F, and (3) O-P-type dimers of mutant GAPDH from B. stearothermophilus with amino acid substitutions Y46G/S48G and Y46G/R52G. It was shown that chemically modified GAPDH and the O-R-type mutant dimers bound to GroEL with 1:1 stoichiometry and dissociation constants K(d) of 0.4 and 0.9 muM, respectively. A striking feature of the resulting complexes with GroEL was their stability in the presence of Mg-ATP. Chemically modified GAPDH and the O-R-type mutant dimers inhibited GroEL-assisted refolding of urea-denatured wild-type GAPDH from B. stearothermophilus but did not affect its spontaneous reactivation. In contrast to the O-R-dimers, the O-P-type mutant dimers neither bound nor affected GroEL-assisted refolding of the wild-type GAPDH. Thus, we suggest that interaction of GroEL with certain types of misfolded proteins can result in the formation of stable complexes and the impairment of chaperonin activity.

Animals↗
PubMed2005

The N-terminal domain of PILB from Neisseria meningitidis is a disulfide reductase that can recycle methionine sulfoxide reductases.

The PilB protein of the Neisseria genus comprises three domains. Two forms have been recently reported to be produced in vivo. One form, containing the three domains, is secreted from the bacterial cytoplasm to the outer membrane, whereas the second form, which is cytoplasmic, only contains the central and the C-terminal domains. The secreted form was shown to be involved in survival under oxidative conditions. Although previous studies indicated that the central and the C-terminal domains display methionine sulfoxide reductase A and B activities, respectively, no function was described so far for the N-terminal domain. In the present study, the N-terminal domain of the PilB of Neisseria meningitidis was produced as a folded entity, and its biochemical and enzymatic properties have been determined. The data show that the N-terminal domain possesses a disulfide redox-active site with a redox potential in the range of that of thioredoxin. Moreover, the N-terminal domain, either as an isolated form or included in PilB, recycles the oxidized forms of the methionine sulfoxide reductases like thioredoxin. These results, which show that the N-terminal domain exhibits a disulfide reductase activity and probably has a thioredoxin-fold, are discussed in relation to its possible functional role in Neisseria.

Amino Acid Sequence↗
PubMed2005

The enzymology and biochemistry of methionine sulfoxide reductases.

The methionine sulfoxide reductase (Msr) family is composed of two structurally unrelated classes of monomeric enzymes named MsrA and MsrB, which display opposite stereo-selectivities towards the sulfoxide function. MsrAs and MsrBs, characterized so far, share the same chemical mechanism implying sulfenic acid chemistry. The mechanism includes three steps with (1) formation of a sulfenic acid intermediate with a concomitant release of 1 mol of methionine per mol of enzyme; (2) formation of an intramonomeric disulfide Msr bond followed by; (3) reduction of the oxidized Msr by thioredoxin (Trx). This scheme is in accordance with the kinetic mechanism of both Msrs which is of ping-pong type. For both Msrs, the reductase step is rate-determining in the process leading to the formation of the disulfide bond. The overall rate-limiting step takes place within the thioredoxin-recycling process, likely being associated with oxidized thioredoxin release. The kinetic data support structural recognition between oxidized Msr and reduced thioredoxin. The active sites of both Msrs are adapted for binding protein-bound methionine sulfoxide (MetSO) more efficiently than free MetSO. About 50% of the MsrBs binds a zinc atom, the location of which is in an opposite direction from the active site. Introducing or removing the zinc binding site modulates the catalytic efficiency of MsrB.

Amino Acid Sequence↗
PubMed2005

Insights into the role of the metal binding site in methionine-R-sulfoxide reductases B.

Methionine sulfoxide reductases B (MsrBs) catalyze the reduction of methionine-R-sulfoxide via a three-step chemical mechanism including a reductase step, formation of an intradisulfide bond followed by a thioredoxin recycling process. Fifty percent of the MsrBs, including the Escherichia coli enzyme, possess a metal binding site composed of two CXXC motifs of unknown function. It is located on the opposite side of the active site. The overexpressed E. coli MsrB tightly binds one atom of zinc/iron. Substitution of the cysteines of E. coli MsrB results in complete loss of bound metal and reductase activity, and leads to a low-structured conformation of the protein as shown by CD, fluorescence, and DSC experiments. Introduction of the two CXXC motifs in Neisseria meningitidis MsrB domain leads to a MsrB that tightly binds one atom of zinc/iron, shows a strongly increased thermal stability and displays a reductase activity similar to that of the wild-type but lacking thioredoxin recycling activity. These results demonstrate the stabilizing effect of the metal and the existence of a preformed metal binding site in the nonbound metal MsrB. The data also indicate that metal binding to N. meningitidis MsrB induces subtle structural modifications, which prevent formation of a competent binary complex between oxidized MsrB and reduced thioredoxin but not between reduced MsrB and substrate. The fact that the E. coli and the N. meningitidis MsrBs exhibit a similar thermal stability suggests the existence of other structural factors in the nonbound metal MsrBs that compensate the metal bound stabilizing effect.

Amino Acid Motifs↗
PubMed2004

Kinetic characterization of the catalytic mechanism of methionine sulfoxide reductase B from Neisseria meningitidis.

Methionine sulfoxide reductases catalyze the thioredoxin-dependent reduction of methionine sulfoxide back to methionine. The methionine sulfoxide reductases family is composed of two structurally unrelated classes of enzymes named MsrA and MsrB, which display opposite stereoselectivities toward the sulfoxide function. Both enzymes are monomeric and share a similar three-step chemical mechanism. First, in the reductase step, a sulfenic acid intermediate is formed with a concomitant release of 1 mol of methionine per mol of enzyme. Then, an intradisulfide bond is formed. Finally, Msrs return back to reduced forms via reduction by thioredoxin. In the present study, it is shown for the Neisseria meningitidis MsrB that (1) the reductase step is rate-determining in the process leading to formation of the disulfide bond and (2) the thioredoxin-recycling process is rate-limiting. Moreover, the data suggest that within the thioredoxin-recycling process, the rate-limiting step takes place after the two-electron chemical exchange and thus is associated with the release of oxidized thioredoxin.

Bacterial Proteins↗
PubMed2004

Evidence for a new sub-class of methionine sulfoxide reductases B with an alternative thioredoxin recognition signature.

Methionine sulfoxide reductases catalyze the reduction of protein-bound methionine sulfoxide back to methionine via a thioredoxin-recycling process. Two classes of methionine sulfoxide reductases, called MsrA and MsrB, exist that display opposite stereoselectivities toward the sulfoxide function. Although they are structurally unrelated, they share a similar chemical mechanism that includes three steps with 1) formation of a sulfenic acid intermediate with a concomitant release of 1 mol of methionine per mole of enzyme; 2) formation of an intradisulfide Msr bond; and 3) reduction of the oxidized Msr by thioredoxin. In the MsrBs that have been biochemically, enzymatically, and structurally characterized so far, the cysteine involved in the regeneration of the catalytic Cys-117 is Cys-63. Cys-117 is located on a beta strand, whereas the recycling Cys-63 is on a loop near Cys-117. The distance between the two cysteines is compatible with formation of the Cys-117/Cys-63 intradisulfide bond. Analyses of MsrB sequences show that at least 37% of the MsrBs do not possess the recycling Cys-63. In the present study, it is shown that Cys-31 in the Xanthomonas campestris MsrB, which is located on another loop, can efficiently substitute for Cys-63. Such a result implies flexibility of the MsrB structures, at least of the loops on which Cys-31 or Cys-63 are located. The fact that about 25% of the putative MsrBs have no recycling cysteine supports other recycling processes in which thioredoxin is not operative.

Amino Acid Sequence↗
PubMed2004

Expression, purification, crystallization and preliminary X-ray diffraction data of methylmalonate-semialdehyde dehydrogenase from Bacillus subtilis.

Methylmalonate-semialdehyde dehydrogenase from Bacillus subtilis was cloned and overexpressed in Escherichia coli. Suitable crystals for X-ray diffraction experiments were obtained by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 195.2, b = 192.5, c = 83.5 A, and contain one tetramer per asymmetric unit. X-ray diffraction data were collected to 2.5 A resolution using a synchrotron-radiation source. The crystal structure was solved by the molecular-replacement method.

Aldehyde Oxidoreductases↗