Function and Structure of a Terpene Synthase Encoded in a Giant Virus Genome

Giant viruses are nonstandard viruses with large particles and genomes. While previous studies have shown that their genomes contain various sequences of interest, their genes related specifically to natural product biosynthesis remain unexplored. Here we analyze the function and structure of a terpene synthase encoded by the gene of a giant virus. The enzyme is phylogenetically separated from the terpene synthases of cellular organisms; however, heterologous gene expression revealed that it still functions as a terpene synthase and produces a cyclic terpene from a farnesyl diphosphate precursor. Crystallographic analysis revealed its protein structure, which is relatively compact but retains essential motifs of the terpene synthases. We thus suggest that like cellular organisms, giant viruses produce and utilize natural products for their ecological strategies

Structure of DesB.

<p>(<b>A</b>) The crystal structure of the DesB dimer. The asymmetric unit of the crystal contains the whole dimer. Subunit A is shown in rainbow colours along the polypeptide chain from the N (blue) to the C (red) terminus. Subunit B is shown in light grey. (<b>B</b>) The Fe (II) coordination sphere of DesB and an anomalous difference Fourier map (contour level of 5.0 σ). (<b>C</b>) The active site of the anaerobic DesB-gallate complex. Carbon atoms of bound gallate are shown in orange. Hydrogen bonds are indicated by pink dotted lines with distances in Å units. (<b>D</b>) Comparison of the coordination spheres between DesB (carbon atoms in green) and LigAB (carbon atoms in yellow) in the substrate (PCA) complex forms <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092249#pone.0092249-Sugimoto1" target="_blank">[9]</a>. Carbon atoms of gallate are shown in orange.</p

Catalytic reaction of DesB.

<p>(<b>A</b>) The chemical structure of gallate and the catalytic reaction of DesB. (<b>B</b>) The chemical structures of PCA and 3MGA.</p

His124 forms a hydrogen bond with a hydroxyl group of gallate and PCA.

<p>(<b>A</b>) Superposition of the active sites of DesB in the substrate-free (white), anaerobic PCA-complex (carbon atoms in yellow) and anaerobic gallate-complex (carbon atoms in green) forms. Carbon atoms of gallate are shown in orange. (<b>B</b>) Crystal preparation scheme of the anaerobic His124Phe DesB-gallate complex (upper panel) and its active site structure with an anomalous difference Fourier map (blue, 5.0 σ) (lower panel). A simulated annealing omit map for gallate (cyan) is also shown (3.0 σ). The thin stick model in pink indicates the position of gallate in the anaerobic DesB-gallate complex.</p

Substrate recognition and Fe (II) shift in the catalytic reaction of DesB.

<p>The light green circle represents the productive binding site for gallate. The pink circle represents the productive coordination sphere with the Fe (II) ion at the A-site.</p

Crystallographic summary of DesB (I).

<p>The highest resolution shell is shown in parenthesis.</p><p>WT: wild type.</p><p>H124F: His124Phe</p>†<p>Protomer A shows substantially higher averaged B-factor (89.2 Ų) than that of protomer B (42.9 Ų). PCA was observed only in protomer B.</p

Molecular Mechanism of Strict Substrate Specificity of an Extradiol Dioxygenase, DesB, Derived from <i>Sphingobium</i> sp. SYK-6

<div><p>DesB, which is derived from <i>Sphingobium</i> sp. SYK-6, is a type II extradiol dioxygenase that catalyzes a ring opening reaction of gallate. While typical extradiol dioxygenases show broad substrate specificity, DesB has strict substrate specificity for gallate. The substrate specificity of DesB seems to be required for the efficient growth of <i>S.</i> sp. SYK-6 using lignin-derived aromatic compounds. Since direct coordination of hydroxyl groups of the substrate to the non-heme iron in the active site is a critical step for the catalytic reaction of the extradiol dioxygenases, the mechanism of the substrate recognition and coordination of DesB was analyzed by biochemical and crystallographic methods. Our study demonstrated that the direct coordination between the non-heme iron and hydroxyl groups of the substrate requires a large shift of the Fe (II) ion in the active site. Mutational analysis revealed that His124 and His192 in the active site are essential to the catalytic reaction of DesB. His124, which interacts with OH (4) of the bound gallate, seems to contribute to proper positioning of the substrate in the active site. His192, which is located close to OH (3) of the gallate, is likely to serve as the catalytic base. Glu377’ interacts with OH (5) of the gallate and seems to play a critical role in the substrate specificity. Our biochemical and structural study showed the substrate recognition and catalytic mechanisms of DesB.</p></div

Active site structures of the DesB-gallate and DesB-PCA complexes.

<p>(<b>A</b>) Crystal preparation scheme of the anaerobic DesB-gallate complex (upper panel) and alternative conformations of the Fe (II) ion observed in the anaerobic DesB-gallate complex (lower panel). An anomalous difference Fourier density (blue) is contoured at the 5.0 σ level. (<b>B</b>) Omit maps for A- and R-site Fe (II) ions (5 σ.. The green and red densities are omit maps for the Fe (II) ions at the A- and R-sites, respectively. (<b>C</b>) Crystal preparation scheme of the aerobic DesB-gallate complex (upper panel) and an anomalous difference Fourier map (5.0 σ) of the aerobic DesB-gallate complex (lower panel). Thin white sticks and small white spheres indicate the positions of gallate and the Fe ion found in the anaerobic DesB-gallate complex, respectively. (<b>D</b>) Crystal preparation scheme of the anaerobic DesB-PCA complex (upper panel) and its active site structure with an anomalous difference Fourier map (5.0 σ) (lower panel). Carbon atoms of PCA are shown in yellow. The thin stick model in orange indicates the position of gallate in the anaerobic DesB-gallate complex.</p

Active site structures of mouse and human SMP30/GNL.

<p>(<b>A</b>) Mouse SMP30/GNL in the substrate-free form, (<b>B</b>) the mouse SMP30/GNL–1,5-AG complex, (<b>C</b>) the human SMP30/GNL–1,5-AG complex, (<b>D</b>) the mouse SMP30/GNL–d-glucose complex, and (<b>E</b>) the mouse SMP30/GNL–xylitol complex. Lid loop residues of mouse SMP30/GNL and human SMP30/GNL are shown in purple and blue, respectively. Carbon atoms of ligand residues for the divalent metal ion (orange sphere) and those for substrate/product analogues are shown in green and yellow, respectively. Other carbon atoms are shown in white.</p

Structural comparison of the lid loops of mouse and human SMP30/GNL.

<p>(<b>A</b>) Lid loops of mouse and human SMP30/GNL in the substrate free form are shown in purple and blue, respectively. The divalent metal ion (labeled as M²⁺) is shown in orange. (<b>B, C</b>) SA-omit maps (mFo-DFc maps) for the lid loop residues in mouse (<b>B</b>) and human (<b>C</b>) SMP30/GNL. The contour levels of the SA-omit maps are 3.0 σ and 2.0 σ for panels B and C, respectively. (<b>D</b>) Surface representation of mouse SMP30/GNL around the lid loop. The entrance for the substrate-binding cavity is indicated by an arrow. Residues in the lid loop are shown in purple.</p