Biomedical subjects

C Corbier

Publications and source records attributed to C Corbier.

16 recordsLinked to original sources

PubMed2001

Crystal structure of the wild-type and D30A mutant thioredoxin h of Chlamydomonas reinhardtii and implications for the catalytic mechanism.

Thioredoxins are ubiquitous proteins which catalyse the reduction of disulphide bridges on target proteins. The catalytic mechanism proceeds via a mixed disulphide intermediate whose breakdown should be enhanced by the involvement of a conserved buried residue, Asp-30, as a base catalyst towards residue Cys-39. We report here the crystal structure of wild-type and D30A mutant thioredoxin h from Chlamydomonas reinhardtii, which constitutes the first crystal structure of a cytosolic thioredoxin isolated from a eukaryotic plant organism. The role of residue Asp-30 in catalysis has been revisited since the distance between the carboxylate OD1 of Asp-30 and the sulphur SG of Cys-39 is too great to support the hypothesis of direct proton transfer. A careful analysis of all available crystal structures reveals that the relative positioning of residues Asp-30 and Cys-39 as well as hydrophobic contacts in the vicinity of residue Asp-30 do not allow a conformational change sufficient to bring the two residues close enough for a direct proton transfer. This suggests that protonation/deprotonation of Cys-39 should be mediated by a water molecule. Molecular-dynamics simulations, carried out either in vacuo or in water, as well as proton-inventory experiments, support this hypothesis. The results are discussed with respect to biochemical and structural data.

Amino Acid Sequence↗

PubMed2001

Biological activities and structural properties of the atypical bacteriocins mesenterocin 52b and leucocin b-ta33a.

The antibacterial spectra and modes of action of synthetic peptides corresponding to mesenterocin 52B and leucocin B-TA33a greatly differ despite their high sequence homology. Circular dichroism experiments establish the capacity of each of these two peptides to partly fold into an amphiphilic helix that might be crucial for their adsorption at lipophilic-hydrophilic interfaces.

Amino Acid Sequence↗

PubMed2000

Piv-Propsi

The pseudodipeptide, (S)-N-isopropyl [N-(pivaloyl)pyrrolidin-2-yl]methylaminooxyacetamide, C(15)H(29)N(3)O(3), adopts a global extended conformation with the hydroxylamine group in the g(+)/g(-) structure. The C-terminal amide NH interacts intramolecularly with the hydroxylamine O atom. Both NH bonds of each molecule are hydrogen bonded to the C-terminal amide carbonyl of a neighbouring molecule.

Journal Article↗

PubMed1997

Comparative enzymatic properties of GapB-encoded erythrose-4-phosphate dehydrogenase of Escherichia coli and phosphorylating glyceraldehyde-3-phosphate dehydrogenase.

GapB-encoded protein of Escherichia coli and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) share more than 40% amino acid identity. Most of the amino acids involved in the binding of cofactor and substrates to GAPDH are conserved in GapB-encoded protein. This enzyme shows an efficient non-phosphorylating erythrose-4-phosphate dehydrogenase activity (Zhao, G., Pease, A. J., Bharani, N., and Winkler, M. E. (1995) J. Bacteriol. 177, 2804-2812) but a low phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity, whereas GAPDH shows a high efficient phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity and a low phosphorylating erythrose-4-phosphate dehydrogenase activity. To identify the structural factors responsible for these differences, comparative kinetic and binding studies have been carried out on both GapB-encoded protein of Escherichia coli and GAPDH of Bacillus stearothermophilus. The KD constant of GapB-encoded protein for NAD is 800-fold higher than that of GAPDH. The chemical mechanism of erythrose 4-phosphate oxidation by GapB-encoded protein is shown to proceed through a two-step mechanism involving covalent intermediates with Cys-149, with rates associated to the acylation and deacylation processes of 280 s-1 and 20 s-1, respectively. No isotopic solvent effect is observed suggesting that the rate-limiting step is not hydrolysis. The rate of oxidation of glyceraldehyde 3-phosphate is 0.12 s-1 and is hydride transfer limiting, at least 2000-fold less efficient compared with that of erythrose 4-phosphate. Thus, it can be concluded that it is only the structure of the substrates that prevails in forming a ternary complex enzyme-NAD-thiohemiacetal productive (or not) for hydride transfer in the acylation step. This conclusion is reinforced by the fact that the rate of oxidation for erythrose 4-phosphate by GAPDH is 0.1 s-1 and is limited by the acylation step, whereas glyceraldehyde 3-phosphate acylation is efficient and is not rate-determining (>/=800 s-1). Substituting Asn for His-176 on GapB-encoded protein, a residue postulated to facilitate hydride transfer as a base catalyst, decreases 40-fold the kcat of glyceraldehyde 3-phosphate oxidation. This suggests that the non-efficient positioning of the C-1 atom of glyceraldehyde 3-phosphate relative to the pyridinium of the cofactor within the ternary complex is responsible for the low catalytic efficiency. No phosphorylating activity on erythrose 4-phosphate with GapB-encoded protein is observed although the Pi site is operative as proven by the oxidative phosphorylation of glyceraldehyde 3-phosphate. Thus the binding of inorganic phosphate to the Pi site likely is not productive for attacking efficiently the thioacyl intermediate formed with erythrose 4-phosphate, whereas a water molecule is an efficient nucleophile for the hydrolysis of the thioacyl intermediate. Compared with glyceraldehyde-3-phosphate dehydrogenase activity, this corresponds to an activation of the deacylation step by >/=4.5 kcal.mol-1. Altogether these results suggest subtle structural differences between the active sites of GAPDH and GapB-encoded protein that could be revealed and/or modulated by the structure of the substrate bound. This also indicates that a protein engineering approach could be used to convert a phosphorylating aldehyde dehydrogenase into an efficient non-phosphorylating one and vice versa.

Aldehyde Oxidoreductases↗

PubMed1996

Comparison of the structures of wild-type and a N313T mutant of Escherichia coli glyceraldehyde 3-phosphate dehydrogenases: implication for NAD binding and cooperativity.

The crystal structure of wild-type and N313T mutant glyceraldehyde 3-phosphate dehydrogenases from Escherichia coli was determined in the presence of NAD at 1.8 angstrom and 2.17 angstrom, respectively. The structure of the monomer and of the tetramer are similar to those observed for other GAPDHs. An exhaustive analysis of the hydrophobic clusters and the hydrogen bond networks explain the high degree of sequence conservation in GAPDHs. The structural effect of the N313T mutation is a change in the (phi,psi) angles of nearby residues Asn236 and Val237, while the structure around the mutated residue remains unchanged. A detailed comparison of the wild-type and N313T mutant E. coli GAPDH with the apo and holo forms of Bacillus stearothermophilus GAPDH is carried out in relation to the apo --> holo transition. An unbiased set of about 60 residues, whose C(alpha) atoms remain in the same relative position in the different forms of the tetramer, is defined as the tetramer "core" which acts as a fixed scaffold around which structural rearrangements occur during the apo --> holo transition. This core essentially includes beta-strands from the beta-sheets forming the O-P and Q-R interfaces, in particular strand beta1 which bears catalytic residue His176. During the apo --> holo transition, dimer O-P rotates around the molecular P-axis by about +1 degrees, and dimer O-R by about -1 degrees. Further rotations of the NAD binding domain relative to the catalytic domain are discussed in relation to the molecular symmetry. The possible effect on NAD binding cooperativity of mutations around the tetramer core is exemplified by residue 252. The presence of a conserved hydrophilic patch embedded in the hydrophobic O-P interface is highlighted. A mechanism for substrate binding, different from those currently proposed, is described where the hydroxyl group of the substrate C(2) atom is hydrogen bonded to Cys149N.

Allosteric Regulation↗

PubMed1995

Crystallization and preliminary X-ray diffraction studies of Escherichia coli glyceraldehyde-3-phosphate dehydrogenase.

Phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in glycolysis. Single crystals of NAD-dependent GAPDH from Escherichia coli have been obtained by vapour diffusion at room temperature using trisodium citrate as precipitant. In almost the same crystallization conditions, two kinds of crystals were found to be suitable for X-ray diffraction. The crystals with only one half of a tetramer in the asymmetric unit were chosen for high- resolution analysis. They belonged to space group C222(1), with cell dimensions a = 79.1, b = 189.6 and c = 122.2 A. These crystals diffracted to 1.8 A resolution.

Journal Article↗

PubMed1995

Circular permutation within the coenzyme binding domain of the tetrameric glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus.

A circularly permuted (cp) variant of the phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus has been constructed with N- and C-termini created within the coenzyme binding domain. The cp variant has a kcat value equal to 40% of the wild-type value, whereas Km and KD values for NAD show a threefold decrease compared to wild type. These results indicate that the folding process and the conformational changes that accompany NAD binding during the catalytic event occur efficiently in the permuted variant and that NAD binding is tighter. Reversible denaturation experiments show that the stability of the variant is only reduced by 0.7 kcal/mol compared to the wild-type enzyme. These experiments confirm and extend results obtained recently on other permuted proteins. For multimeric proteins, such as GAPDH, which harbor subunits with two structural domains, the natural location of the N- and C-termini is not a prerequisite for optimal folding and biological activity.

Amino Acid Sequence↗

PubMed1994

Characterization of the two anion-recognition sites of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus by site-directed mutagenesis and chemical modification.

The active site of the glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) contains two anion recognition sites which have been attributed to the phosphate binding of the substrates, namely, glyceraldehyde 3-phosphate (Ps site) and inorganic phosphate (Pi site) [Moras et al. (1975) J. Biol. Chem. 250, 9137-9162]. In order to probe the role of both sites during the catalytic event, Arg 195 from the Pi site and Arg 231 from the Ps site of the Bacillus stearothermophilus enzyme have been changed to Leu and Gly, respectively, by site-directed mutagenesis. A comparative study of the chemical reactivity of the mutants and wild type toward 2,3-butanedione revealed a similarly high reactivity only for the R195L mutant and wild type, suggesting that only Arg 231 is chemically reactive toward 2,3-butanedione and that its reactivity is not influenced by the presence of the residue Arg 195, which is only 4 A distant. The kinetic consequences of the mutations were also analyzed for the consecutive steps in the forward catalytic reaction. The replacement of Arg 195 by Leu leads to a marked decrease of the rate of the first steps of the reaction which lead to the acylenzyme formation, in particular, the rate of enzyme-substrate association, while these steps occur at a similar or higher rate when Arg 231 is replaced by Gly. Furthermore, the mutations R195L and R231G also result in a 550-fold and 16,400-fold decrease in the second-order rate constant of phosphorolysis. This step becomes rate-determining for the R195L mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

Anions↗

PubMed1993

Determinants of coenzyme specificity in glyceraldehyde-3-phosphate dehydrogenase: role of the acidic residue in the fingerprint region of the nucleotide binding fold.

On the basis of the three-dimensional structure of the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and of sequence comparison with the photosynthetic NAD(P)-dependent GAPDH of the chloroplast, a series of mutants of GAPDH from Bacillus stearothermophilus have been constructed. The results deduced from kinetic and binding studies suggest that the absence of activity of the wild-type GAPDH with NADP as a cofactor is the consequence of at least three factors: (1) steric hindrance, (2) electrostatic repulsion between the charged carboxyl group of Asp32 and the 2'PO4, and (3) structural determinants at the subunit interface of the tetramer. The best value for kcat/KM and KD for NADP was observed for the D32A-L187A-P188S mutant. This triple mutation leads to a switch in favor of NADP specificity but with a kcat/KM ratio 50- and 80-fold less than that observed for the wild type with NAD and for the chloroplast GAPDH with NADP, respectively. Substituting the invariant chloroplastic Thr33-Gly34-Gly35 for the B. stearothermophilus Leu33-Thr34-Asp35 residues on the double mutant Ala187-Ser188 does not improve significantly the affinity for NADP while substituting Ala32 for Asp32 on the double mutant does. Clearly, other subtle adjustments in the adenosine subsite are needed to reconcile the presence of the carboxylate group of Asp32 and the 2'-phosphate of NADP. Kinetic studies indicate a change of the rate-limiting step for the mutants. This could be the consequence of an incomplete apo-holo transition.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

A new chemical mechanism catalyzed by a mutated aldehyde dehydrogenase.

NAD(P) aldehyde dehydrogenases (EC 1.2.1.3) are a family of enzymes that oxidize a wide variety of aldehydes into acid or activated acid compounds. Using site-directed mutagenesis, the essential nucleophilic Cys 149 in the NAD-dependent phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Escherichia coli has been replaced by alanine. Not unexpectedly, the resulting mutant no longer shows any oxidoreduction phosphorylating activity. The same mutation, however, endows the enzyme with a novel oxidoreduction nonphosphorylating activity, converting glyceraldehyde 3-phosphate into 3-phosphoglycerate. Our study further provides evidence for an alternative mechanism in which the true substrate is the gem-diol entity instead of the aldehyde form. This implies that no acylenzyme intermediate is formed during the catalytic event. Therefore, the mutant C149A is a new enzyme which catalyzes a distinct reaction with a chemical mechanism different from that of its parent phosphorylating glyceraldehyde-3-phosphate dehydrogenase. This finding demonstrates the possibility of an alternative route for the chemical reaction catalyzed by classical nonphosphorylating aldehyde dehydrogenases.

Catalysis↗

PubMed1990

Probing the coenzyme specificity of glyceraldehyde-3-phosphate dehydrogenases by site-directed mutagenesis.

By combining our knowledge of the crystal structure of the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the sequence of the photosynthetic NADP-dependent GAPDH of the chloroplast, two particular amino acid residues were predicted as the principal determinants of differing coenzyme specificity. By use of site-directed mutagenesis, the amino acids Leu 187 and Pro 188 of GAPDH from Bacillus stearothermophilus have been replaced with Ala 187 and Ser 188, which occur in the sequence from the chloroplast enzyme. The resulting mutant was shown to be catalytically active not only with its natural coenzyme NAD but also with NADP, thus confirming the initial hypothesis. This approach has not only enabled us to alter the coenzyme specificity by minimal amino acid changes but also revealed factors that control the relative affinity of the enzyme for NAD and NADP.

Amino Acid Sequence↗

PubMed1990

The nicotinamide subsite of glyceraldehyde-3-phosphate dehydrogenase studied by site-directed mutagenesis.

Directed mutagenesis has been used to study the nicotinamide subsite of the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Residue Asn313 is involved together with the carboxyamide moiety of the nicotinamide ring in a complex network of hydrogen bonding interactions which fix the position of the pyridinium ring of NAD to which hydride transfer occurs at the C-4 position in the catalytic reaction. The asparagine side-chain has been replaced by that of the Thr and Ala residues and results in mutants with very similar properties. Both mutants show much weaker binding of NAD and lower catalytic efficiency. The mutant Asn313----Thr still exhibits strict B-stereospecificity in hydride transfer and retains the property of negative co-operativity in NAD binding. These experiments strongly suggest that the mutant enzyme undergoes the apo----holo sub-unit structural transition associated with coenzyme binding but that the nicotinamide ring is no longer as rigidly held in its pocket as in the wild type enzyme. The results shed light on the details of the molecular interactions which are responsible for negative co-operativity in this enzyme.

Apoenzymes↗

PubMed1989

Role of the histidine 176 residue in glyceraldehyde-3-phosphate dehydrogenase as probed by site-directed mutagenesis.

The catalytically essential amino acid, histidine 176, in the active site of Escherichia coli glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been replaced with an asparagine residue by site-directed mutagenesis. The role of histidine 176 as a chemical activator, enhancing the reactivity of the thiol group of cysteine 149, has been demonstrated, with iodoacetamide as a probe. The esterolytic properties of GAPDH, illustrated by the hydrolysis of p-nitrophenyl acetate, have been also studied. The kinetic results favor a role for histidine 176 not only as a chemical activator of cysteine 149 but also as a hydrogen donor facilitating the formation of tetrahedral intermediates. These results support the hypothesis that histidine 176 plays a similar role during the oxidative phosphorylation of glyceraldehyde 3-phosphate.

Binding Sites↗

PubMed1989

Site-directed mutagenesis of glyceraldehyde-3-phosphate dehydrogenase reveals the role of residue Ser148.

A mutant of Bacillus stearothermophilus D-glyceraldehyde-3-phosphate dehydrogenase, Ser148----Ala, was produced by oligonucleotide-directed mutagenesis. The study of the catalytic properties of this mutant has shown that this mutation significantly affects the Michaelis constant of inorganic phosphate and to a lesser extent that of 1,3-diphosphoglycerate and D-glyceraldehyde-3-phosphate. This result is consistent with model-building studies which show that, for the phosphorylation step of catalysis, inorganic phosphate must bind to the anion recognition site designated Pi with the C(3) phosphate of the acyl-enzyme intermediate in the alternative anion site Ps. Studies of the enantiomeric specificity using D- and L-glyceraldehyde as substrates show that the hydroxyl group of Ser148, combined with the presence of the C(3) phosphate of the substrate, enhances stereospecificity as well as catalysis. However, the stereospecific effect cannot be a consequence of the direct interaction of Ser148 with the C(2)-hydroxyl of the substrate. The changed Km for glyceraldehyde-3-phosphate suggests that the initial step of hemithioacetal formation may take place with its C(3) phosphate bound in the Pi site. This supports the molecular mechanism proposed by Moody (1984). Therefore, catalysis could be enhanced through interactions of the serine hydroxyl group not only with inorganic phosphate but also with the C(3) phosphate of glyceraldehyde-3-phosphate.

Binding Sites↗

PubMed1988

Use of site-directed mutagenesis to probe the role of Cys149 in the formation of charge-transfer transition in glyceraldehyde-3-phosphate dehydrogenase.

Oligonucleotide-directed mutagenesis was employed to produce mutants of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Escherichia coli and Bacillus stearothermophilus. Three different mutants proteins--His176----Asn, Cys149----Ser, Cys149----Gly--were isolated from one or both of the enzymes. The study of the properties of these mutants has shown that Cys149 is clearly responsible for the information of a charge-transfer transition, named the Racker band, observed during the NAD+ binding to apoGAPDH. This result excludes a similarity between the Racker band and the charge-transfer transition observed following the alkylation of GAPDH by 3-chloroacetyl pyridine-adenine dinucleotide.

Binding Sites↗

PubMedDate not supplied

Crystal structure of the W35A mutant thioredoxin h from Chlamydomonas reinhardtii: the substitution of the conserved active site Trp leads to modifications in the environment of the two catalytic cysteines.

The conformational analysis of W35A thioredoxin h from the eukaryotic green alga Chlamydomonas reinhardtii in the solid state has been carried out by x-ray diffraction, with the aim to clarify the role of Trp in the catalysis. Comparative analysis of W35A mutant with wild-type (WT) thioredoxin shows that, even if the structural motif of thioredoxin is not perturbed, the substitution of Trp35 by an Ala leads to significant changes in protein conformation near the active site. This rearrangement increases its solvent exposure and explains the change of the pKa values of the catalytic cysteines. The substitution of the Trp residue also influences the crystal packing as well as the recognition ability of thioredoxin. The solid state analysis suggests that the Trp residue has a structural function both to force the active site in the bioactive conformation, and to mediate the protein-protein recognition.

Amino Acid Substitution↗