Journal: PLoS ONE

Article Title: Structural Basis for Binding of Fluorinated Glucose and Galactose to Trametes multicolor Pyranose 2-Oxidase Variants with Improved Galactose Conversion

doi: 10.1371/journal.pone.0086736

Figure Lengend Snippet: Structural overlay of the active sites in Tm 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 3F Glc in the productive 2-oxidation binding mode. The sugar is stabilized as the β -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 . (b) 2F Glc in the competing 3-oxidation binding mode. In H450G, the C1 hydroxyl in 2F Glc is stabilized in axial configuration ( α -anomer) by Asp452 and Thr169 and the substrate-binding loop assumes the semi-open conformation. V546C stabilizes the β -anomer and reveals the open conformation of the substrate-binding loop as observed earlier for H167A in complex with 2F Glc . (c) 3F 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) 2F Gal in competing binding modes. The competing binding mode observed for V546C corresponds to the 2F Gal β -anomer oriented for oxidation at C1. The competing binding mode for H450G shows the α- anomer of 2F 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 away 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 .

Article Snippet: LLC., USA; Cat. N o . F5006-25 mg); 3F Gal (3-deoxy-3-fluoro-D-galactose; Carbosynth Ltd., Newbury, UK; Cat. N o . MD05336); and 2F Gal (2-deoxy-2-fluoro-D-galactose; Toronto Research Chemicals Inc.; Cat. N o . D233000).

Techniques: Mutagenesis, Binding Assay, Produced

Journal: PLoS ONE

Article Title: Structural Basis for Binding of Fluorinated Glucose and Galactose to Trametes multicolor Pyranose 2-Oxidase Variants with Improved Galactose Conversion

doi: 10.1371/journal.pone.0086736

Figure Lengend Snippet: Superposition of mutant structures emphasizing the conformation of the substrate-binding loop. The FAD molecule and the pyranose sugar are shown as ball-and-stick models. (a) Tm P2O variants complexed with 3F Glc corresponding to the productive 2-oxidation binding mode with the substrate-binding loop in the semi-open conformation. The relaxation induced by the H450G replacement (green and light-blue models) is highlighted by a shaded circle. The H167A model corresponds to PDB code 3PL8 . (b) Mutant complexes with bound 2F Glc in the competing 3-oxidation binding mode. The H167A (PDB code 2IGO ) and V546C variants show the open loop conformation, which also has been observed for the wild type. H450G and H450G/V546C show the productive semi-open loop conformation. (c) Mutant complexes of V546C, H450G and H450G/V546C with bound 3F Gal in the productive 2-oxidation binding mode with the substrate-binding loop in the semi-open conformation. The wild-type mimic H167A displays did not bind the sugar and displays the closed, occluded loop conformation that is typically observed for Tm P2O in the absence of oxidizable sugar. The closed loop conformation is incompatible with sugar binding. (d) Mutant complexes with bound 2F Gal. Despite the fundamentally different competing modes observed for H167A and V546C (C1-oxdiation mode) and H450G and H450G/V546C (C3-oxidation mode), all complexes show the substrate-binding loop in the semi-open loop conformation associated with productive sugar binding. The pictures were produced using the program PyMOL .

Article Snippet: LLC., USA; Cat. N o . F5006-25 mg); 3F Gal (3-deoxy-3-fluoro-D-galactose; Carbosynth Ltd., Newbury, UK; Cat. N o . MD05336); and 2F Gal (2-deoxy-2-fluoro-D-galactose; Toronto Research Chemicals Inc.; Cat. N o . D233000).

Techniques: Mutagenesis, Binding Assay, Produced