41 research outputs found

    Structure of SePSK in complex with AMP-PNP.

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    <p>(A) The electron density of AMP-PNP. The SePSK structure is shown in the electrostatic potential surface mode. The AMP-PNP is depicted as sticks with its <i>ǀFoǀ-ǀFcǀ</i> map contoured at 3 σ shown as cyan mesh. (B) The AMP-PNP binding pocket. The head of AMP-PNP is sandwiched by four residues (Leu293, Gly376, Gly377 and Trp383). The protein skeleton is shown as cartoon (cyan). The four α-helices (α26, α28, α27 and α30) are labeled in red. The AMP-PNP and coordinated residues are shown as sticks. The interactions between them are represented as black dashed lines. The numerical note near the black dashed line indicates the distance (Å).</p

    The binding of D-ribulose (RBL) with SePSK.

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    <p>(A) The electrostatic potential surface map of RBL-SePSK and a zoom-in view of RBL binding site. The RBL1 and RBL2 are depicted as sticks. (B) Interaction of two D-ribulose molecules (RBL1 and RBL2) with SePSK. The RBL molecules (carbon atoms colored yellow) and amino acid residues of SePSK (carbon atoms colored green) involved in RBL interaction are shown as sticks. The hydrogen bonds are indicated by the black dashed lines and the numbers near the dashed lines are the distances (Å). (C) The binding affinity assays of SePSK with D-ribulose. Single-cycle kinetic data are reflecting the interaction of SePSK and D8A-SePSK with D-ribulose. It shows two experimental sensorgrams after minus the empty sensorgrams. The original data is shown as black curve, and the fitted data is shown as different color (wild type SePSK: red curve, D8A-SePSK: green curve). Dissociation rate constant of wild type and D8A-SePSK are 3 ms<sup>-1</sup> and 9 ms<sup>-1</sup>, respectively.</p

    Simulated conformational change of SePSK during the catalytic process.

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    <p>The structures are shown as cartoon and the ligands are shown as sticks. Domain I from D-ribulose-SePSK (green) and Domain II from AMP-PNP-SePSK (cyan) are superposed with apo-AtXK-1 (1<sup>st</sup>), apo-SePSK (2<sup>nd</sup>), 3LL3 (3<sup>rd</sup>) and 1GLJ (4<sup>th</sup>), respectively. The numbers near the black dashed lines show the distances (Å) between two nearest atoms of RBL and AMP-PNP.</p

    Crystal Structures of Putative Sugar Kinases from <i>Synechococcus Elongatus</i> PCC 7942 and <i>Arabidopsis Thaliana</i>

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    <div><p>The genome of the <i>Synechococcus elongatus</i> strain PCC 7942 encodes a putative sugar kinase (SePSK), which shares 44.9% sequence identity with the xylulose kinase-1 (AtXK-1) from <i>Arabidopsis thaliana</i>. Sequence alignment suggests that both kinases belong to the ribulokinase-like carbohydrate kinases, a sub-family of FGGY family carbohydrate kinases. However, their exact physiological function and real substrates remain unknown. Here we solved the structures of SePSK and AtXK-1 in both their apo forms and in complex with nucleotide substrates. The two kinases exhibit nearly identical overall architecture, with both kinases possessing ATP hydrolysis activity in the absence of substrates. In addition, our enzymatic assays suggested that SePSK has the capability to phosphorylate D-ribulose. In order to understand the catalytic mechanism of SePSK, we solved the structure of SePSK in complex with D-ribulose and found two potential substrate binding pockets in SePSK. Using mutation and activity analysis, we further verified the key residues important for its catalytic activity. Moreover, our structural comparison with other family members suggests that there are major conformational changes in SePSK upon substrate binding, facilitating the catalytic process. Together, these results provide important information for a more detailed understanding of the cofactor and substrate binding mode as well as the catalytic mechanism of SePSK, and possible similarities with its plant homologue AtXK-1.</p></div

    Overall structures of SePSK and AtXK-1.

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    <p>(A) Three-dimensional structure of apo-SePSK. The secondary structural elements are indicated (α-helix: cyan, β-sheet: yellow). (B) Three-dimensional structure of apo-AtXK-1. The secondary structural elements are indicated (α-helix: green, β-sheet: wheat).</p

    The enzymatic activity assays of SePSK and AtXK-1.

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    <p>(A) The ATP hydrolysis activity of SePSK and AtXK-1. Both SePSK and AtXK-1 showed ATP hydrolysis activity in the absence of substrate. While the ATP hydrolysis activity of SePSK greatly increases upon addition of D-ribulose (DR). (B) The ATP hydrolysis activity of SePSK with addition of five different substrates. The substrates are DR (D-ribulose), LR (L-ribulose), DX (D-xylulose), LX (L-xylulose) and GLY (Glycerol). (C) The ATP hydrolysis activity of SePSK and AtXK-1 with or without D-ribulose. (D) The ATP hydrolysis activity of wild-type (WT) and single-site mutants of SePSK. Three single-site mutants of SePSK are D8A-SePSK, T11A-SePSK and D221A-SePSK. The ATP hydrolysis activity measured via luminescent ADP-Glo assay (Promega).</p

    Positions of the positive mutation sites in AFEST.

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    <p>Mutated residues are marked in green and catalytic residues of AFEST are marked in purple.</p

    Comparison of volume uniformity of the internal water phase of w/o/w double emulsion droplets generated by membrane-extrusion (A) and homogenizing (B).

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    <p>Different-colored peaks represent different concentrations of <i>7-Hydroxycoumarin-3-carboxylic acid</i> in the internal water phase: (red) 10 µM, (blue) 100 µM, (green) 1000 µM.</p

    Model screening of AFEST-displaying cells.

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    <p>Model screening of AFEST-displaying cells.</p

    Comparison among three different w/o/w double emulsion droplet generation methods.

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    <p>Comparison among three different w/o/w double emulsion droplet generation methods.</p
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