Principal productive and competing binding modes for fluorinated glucose and galactose.

Abstract

<p>Structural overlay of the active sites in <i>Tm</i>P2O V546C (beige) and H450G (green). The V546C mutant is used as a reference since it displays the same binding modes as the H167A mutant, and for 2- or 3-fluorinated glucose, also agrees with the binding modes observed for the wild type. (a) Binding of <i>3F</i>Glc in the productive 2-oxidation binding mode. The sugar is stabilized as the <i>β</i>-anomer with O2 coordinated by His458 and Asn593 and C2 appropriately positioned for oxidation. The substrate-binding loop is in the semi-open conformation positioning Phe454 closely packed against the pyranose as has been described for the productive binding mode earlier <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086736#pone.0086736-Tan1" target="_blank">[19]</a>. (b) <i>2F</i>Glc in the competing 3-oxidation binding mode. In H450G, the C1 hydroxyl in <i>2F</i>Glc is stabilized in axial configuration (<i>α</i>-anomer) by Asp452 and Thr169 and the substrate-binding loop assumes the semi-open conformation. V546C stabilizes the <i>β</i>-anomer and reveals the open conformation of the substrate-binding loop as observed earlier for H167A in complex with <i>2F</i>Glc <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086736#pone.0086736-Kujawa1" target="_blank">[14]</a>. (c) <i>3F</i>Gal in productive 2-oxidation binding mode with the axial C4 hydroxyl group coordinated by Asp452 and Thr169. The substrate-binding loop is in the semi-open conformation. (d) <i>2F</i>Gal in competing binding modes. The competing binding mode observed for V546C corresponds to the <i>2F</i>Gal <i>β</i>-anomer oriented for oxidation at C1. The competing binding mode for H450G shows the <i>α-</i>anomer of <i>2F</i>Gal oriented for oxidation at C3. In both cases, the substrate-binding loop assumes the semi-open conformation compatible with the productive sugar-oxidation mode. All structures that bind sugar substrate show the Thr169 Oγ1 atom pointing <i>away</i> from the flavin N(5)/O(4) locus, which constitutes an additional hallmark of the productive binding mode. For clarity, the covalent link between the flavin and His167 is not shown in the pictures. The pictures were produced using the program PyMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086736#pone.0086736-DeLano1" target="_blank">[43]</a>.</p

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