Etude structurale et fonctionnelle des ADN glucosyltransférases du bactériophage T4

Les deux ADN glucosyltransférases du bactériophage T4 transfèrent le glucose de l'UDP-glucose sur la 5-hydroxyméthylcytosine de l'ADN du phage. L'alpha-glucosyltransférase (AGT) et la beta-glucosyltransférase (BGT) créent respectivement des liaisons alpha- et beta-glucosidiques. Elles appartiennent à la grande famille des glycosyltransférases.L'étude cristallographique exhaustive de BGT a permis d'obtenir des structures correspondant à la plupart des étapes du cycle catalytique de BGT. La structure de BGT en complexe avec le donneur de glucose, l'UDP-glucose, explique la spécificité de l'enzyme à l'égard du glucose. Associée à des études biochimiques, l'analyse structurale démontre un mécanisme réactionnel par transfert en ligne du glucose et identifie la base catalytique. La structure d'AGT révèle un repliement voisin de celui de BGT mais un site de fixation de l'ADN accepteur tout-à-fait différent et d'ailleurs unique chez les glycosyltransférases.Les structures de ces deux enzymes complexées à l'ADN montrent qu'elles utilisent un mécanisme par retournement de base, comme d'autres enzymes de modification de l'ADN. Nous avons pu de surcroît établir que ce processus est passif.The two bacteriophage T4 DNA glucosyltransferases transfer the glucose moiety of UDP-glucose to the 5-hydroxymethylcytosine bases of phage DNA duplex. alpha-glucosyltransferase (AGT) and beta-glucosyltransferase (BGT) make alpha- and beta-glucosidic bond respectively. They belong to the large family of glycosyltransferases.The crystallographic study of BGT allowed us to solve structures showing different steps of the catalytic cycle. The structure of BGT in complex with the sugar donor UDP-glucose explains the specificity towards the glucose. Combined with biochemical data, the structural analyses support an in-line displacement reaction mechanism and identify the catalytic base. The structure of AGT reveals a similar fold as BGT but a different sugar acceptor DNA binding mode that is unique among glycosyltransferases.The DNA complexes with AGT and BGT showed that both enzymes use a base-flipping mechanism, as for other DNA modifying enzymes. Furthermore, we clearly provide evidence for a passive process for both enzymes.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

X-ray structure of a domain-swapped dimer of Ser46-phosphorylated Crh from Bacillus subtilis

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X-ray structure of a bifunctional protein kinase in complex with its protein substrate HPr

HPr kinase/phosphorylase (HprK/P) controls the phosphorylation state of the phosphocarrier protein HPr and regulates the utilization of carbon sources by Gram-positive bacteria. It catalyzes both the ATP-dependent phosphorylation of Ser-46 of HPr and its dephosphorylation by phosphorolysis. The latter reaction uses inorganic phosphate as substrate and produces pyrophosphate. We present here two crystal structures of a complex of the catalytic domain of Lactobacillus casei HprK/P with Bacillus subtilis HPr, both at 2.8-Å resolution. One of the structures was obtained in the presence of excess pyrophosphate, reversing the phosphorolysis reaction and contains serine-phosphorylated HPr. The complex has six HPr molecules bound to the hexameric kinase. Two adjacent enzyme subunits are in contact with each HPr molecule, one through its active site and the other through its C-terminal helix. In the complex with serine-phosphorylated HPr, a phosphate ion is in a position to perform a nucleophilic attack on the phosphoserine. Although the mechanism of the phosphorylation reaction resembles that of eukaryotic protein kinases, the dephosphorylation by inorganic phosphate is unique to the HprK/P family of kinases. This study provides the structure of a protein kinase in complex with its protein substrate, giving insights into the chemistry of the phospho-transfer reactions in both directions

Structural analysis of the bacterial HPr kinase/phosphorylase V267F mutant gives insights into the allosteric regulation mechanism of this bifunctional enzyme.

The HPr kinase/phosphorylase (HPrK/P) is a bifunctional enzyme that controls the phosphorylation state of the phosphocarrier protein HPr, which regulates the utilization of carbon sources in gram-positive bacteria. It uses ATP or pyrophosphate for the phosphorylation of serine 46 of HPr and inorganic phosphate for the dephosphorylation of P-Ser46-HPr via a phosphorolysis reaction. HPrK/P is a hexameric protein kinase of a new type with a catalytic core belonging to the family of P-loop proteins. It exhibits no structural similarity to eukaryotic protein kinases. So far, HPrK/P structures have shown the enzyme in its phosphorylase conformation. They permitted a detailed characterization of the phosphorolysis mechanism. In the absence of a structure with bound nucleotide, we used the V267F mutant enzyme to assess the kinase conformation. Indeed, the V267F replacement was found to cause an almost entire loss of the phosphorylase activity of Lactobacillus casei HPrK/P. In contrast, the kinase activity remained conserved. To elucidate the structural alterations leading to this drastic change of activity, the X-ray structure of the catalytic domain of L. casei HPrK/P-V267F was determined at 2.6 A resolution. A comparison with the structure of the wild type enzyme showed that the mutation induces conformation changes compatible with the switch from phosphorylase to kinase function. Together with nucleotide-binding fluorescence measurements, these results allowed us to decipher the cooperative behavior of the protein and to gain new insights into the allosteric regulation mechanism of HPrK/P

The crystal structure of the human DNA repair endonuclease HAP1 suggests the recognition of extra-helical deoxyribose at DNA abasic sites

The structure of the major human apurinic/ apyrimidinic endonuclease (HAP1) has been solved at 2.2 A resolution. The enzyme consists of two symmetrically related domains of similar topology and has significant structural similarity to both bovine DNase I and its Escherichia coli homologue exonuclease III (EXOIII). A structural comparison of these enzymes reveals three loop regions specific to HAP1 and EXOIII. These loop regions apparently act in DNA abasic site (AP) recognition and cleavage since DNase I, which lacks these loops, correspondingly lacks AP site specificity. The HAP1 structure furthermore suggests a mechanism for AP site binding which involves the recognition of the deoxyribose moiety in an extrahelical conformation, rather than a 'flipped-out' base opposite the AP site