Photoinduced electron transfer in singly labeled thiouredopyrenetrisulfonate azurin derivatives

AbstractA novel method for the initiation of intramolecular electron transfer reactions in azurin is reported. The method is based on laser photoexcitation of covalently attached thiouredopyrenetrisulfonate (TUPS), the reaction that generates the low potential triplet state of the dye with high quantum efficiency. TUPS derivatives of azurin, singly labeled at specific lysine residues, were prepared and purified to homogeneity by ion exchange HPLC. Transient absorption spectroscopy was used to directly monitor the rates of the electron transfer reaction from the photoexcited triplet state of TUPS to Cu(II) and the back reaction from Cu(I) to the oxidized dye. For all singly labeled derivatives, the rate constants of copper ion reduction were one or two orders of magnitude larger than for its reoxidation, consistent with the larger thermodynamic driving force for the former process. Using 3-D coordinates of the crystal structure of Pseudomonas aeruginosa azurin and molecular structure calculation of the TUPS modified proteins, electron transfer pathways were calculated. Analysis of the results revealed a good correlation between separation distance from donor to Cu ligating atom (His-N or Cys-S) and the observed rate constants of Cu(II) reduction

7th Drug hypersensitivity meeting: part two

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Crystal structure of the covalent intermediate of amylosucrase from Neisseria polysaccharea

International audienc

Crystal structure of sucrose phosphorylase from Bifidobacterium adolescentis.

Around 80 enzymes are implicated in the generic starch and sucrose pathways. One of these enzymes is sucrose phosphorylase, which reversibly catalyzes the conversion of sucrose and orthophosphate to d-Fructose and a-d-glucose 1-phosphate. Here, we present the crystal structure of sucrose phosphorylase from Bifidobacterium adolescentis (BiSP) refined at 1.77 Å resolution. It represents the first 3D structure of a sucrose phosphorylase and is the first structure of a phosphate-dependent enzyme from the glycoside hydrolase family 13. The structure of BiSP is composed of the four domains A, B, B‘, and C. Domain A comprises the (ß/a)8-barrel common to family 13. The catalytic active-site residues (Asp192 and Glu232) are located at the tips of ß-sheets 4 and 5 in the (ß/a)8-barrel, as required for family 13 members. The topology of the B‘ domain disfavors oligosaccharide binding and reduces the size of the substrate access channel compared to other family 13 members, underlining the role of this domain in modulating the function of these enzymes. It is remarkable that the fold of the C domain is not observed in any other known hydrolases of family 13. BiSP was found as a homodimer in the crystal, and a dimer contact surface area of 960 Å2 per monomer was calculated. The majority of the interactions are confined to the two B domains, but interactions between the loop 8 regions of the two barrels are also observed. This results in a large cavity in the dimer, including the entrance to the two active sites

Crystal structure of sucrose phosphorylase from Bifidobacterium adolescentis

Around 80 enzymes are implicated in the generic starch and sucrose pathways. One of these enzymes is sucrose phosphorylase, which reversibly catalyzes the conversion of sucrose and orthophosphate to d Fructose and amp; 945; d glucose 1 phosphate. Here, we present the crystal structure of sucrose phosphorylase from Bifidobacterium adolescentis BiSP refined at 1.77 resolution. It represents the first 3D structure of a sucrose phosphorylase and is the first structure of a phosphate dependent enzyme from the glycoside hydrolase family 13. The structure of BiSP is composed of the four domains A, B, B , and C. Domain A comprises the amp; 946; amp; 945; 8 barrel common to family 13. The catalytic active site residues Asp192 and Glu232 are located at the tips of amp; 946; sheets 4 and 5 in the amp; 946; amp; 945; 8 barrel, as required for family 13 members. The topology of the B domain disfavors oligosaccharide binding and reduces the size of the substrate access channel compared to other family 13 members, underlining the role of this domain in modulating the function of these enzymes. It is remarkable that the fold of the C domain is not observed in any other known hydrolases of family 13. BiSP was found as a homodimer in the crystal, and a dimer contact surface area of 960 2 per monomer was calculated. The majority of the interactions are confined to the two B domains, but interactions between the loop 8 regions of the two barrels are also observed. This results in a large cavity in the dimer, including the entrance to the two active site

crystal structures of amylosucrase from Neisseria polysaccharea in complex with D-glucose and the active site mutant Glu328Glm in complex with the natural substrate sucrose

International audienc