Additional file 8: Table S12. of Sequencing and functional annotation of the whole genome of the filamentous fungus Aspergillus westerdijkiae

Structural and functional annotations of the OTA biosynthesis-related clusters on scaffold14 of A. westerdijkiae. Table S13. Structural and functional annotations of the OTA biosynthesis-related cluster on scaffold45 of A. westerdijkiae. (DOCX 153 kb

The inactive tDGC dimer (a) The two monomers present in the asymmetric unit are colored in grey and yellow respectively with the two bridging c-di-GMP molecules shown as sticks.

<p>The views are along (left) and perpendicular (right) to the non-crystallographic dyad. (<b>b</b>) Stereoview of the dimerization mediated by two c-di-GMP molecules (labeled c-di-GMP1 and c-di-GMP2), bound at the I-site of tDGC, forcing the GGDEF domain in an inhibited conformation, with both A-sites facing away from each other. An omit map (blue mesh) with Fourier coefficients 2F_o-F_c, where the c-di-GMP ligand was omitted from phase calculation is shown at 1σ contour level. Residues from the RxxD motif at the I-site forming hydrogen bonds with the bound c-di-GMP molecules are displayed as sticks and the distances between interacting atoms are displayed. The same color code as in panel <b>a</b> is used.</p

Domain architecture of tDGC.

<p>(<b>a</b>) Topology of the GGDEF domain of tDGC. The N- and C- termini of the polypeptide chain are indicated. The β-strands are depicted by pink arrows and α-helices by blue tubes. (<b>b</b>) Location of the A- and I-site on the structure of tDGC: The loops bearing the “GGDEF” motif at the A-site and “RxxD” motif at the I-site are shown in red and blue respectively. (<b>c</b>) Superposition of the A site of tDGC (cyan) and PleD (dark blue)⁷. GTPαS-Mg²⁺, bound to PleD is shown as sticks and green spheres respectively. Residues involved in metal ion binding and base recognition (represented as sticks and labeled according to tDGC and PleD numbering schemes) are strictly conserved between the two proteins.</p

Data collection statistics.

a<p>The values for the highest resolution shell are shown in parenthesis.</p><p>Data collection statistics.</p

The active-like tDGC dimer and the cyclization reaction.

<p>(<b>a</b>) Structure of tDGC crystallized in a dimeric active-like conformation, with the two half active sites (loops containing the GGDEF motif colored in blue) facing each other. One monomer is colored yellow and the other grey. The single c-di-GMP molecule bound to the I-site of the monomer colored in yellow, is shown as sticks. (<b>b</b>) “Optimized” dimer where the residual transformation (<b>Figure S2</b> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110912#pone.0110912.s001" target="_blank">File S1</a>) was applied to the monomer colored grey to generate a dimer with exact two-fold symmetry. The view is along the non-crystallographic dyad that runs across the two GTP molecules displayed as sticks. (<b>c</b>) Magnified view of the tDGC-GTP-Mg Michaelis complex, modeled on the basis of the “optimized” 2-fold symmetric dimer. The arrows indicate the nucleophilic attack of the 3′ oxygen atom on the α-phosphate of the adjacent GTP. (<b>d</b>) Superposition of the “optimized” active dimer (this work, yellow and grey) and the c-diGMP cross-linked YdeH dimer (PDB code: 3TVK, Zaehringer and Schirmer) (colored in pink and teal).</p

Size Exclusion Chromatograms (a) Gel filtration chromatogram using a S75 column, of the wild type tDGC protein (red) that elutes as a c-di-GMP-bound dimer and of the R158A mutant (green) that purifies as a monomer and is devoid of nucleotides.

<p>(b) Following incubation of the wild type tDGC bound to c-di-GMP with RocR (in the gel filtration buffer containing 25 mM Tris-HCl, 300 mM NaCl, 5% (v/v) glycerol, 10 mM MgCl₂ for 2 hrs at room temperature), c-diGMP is converted to pGpG and tDGC elutes predominantly as a monomer. SDS PAGE analysis of the dimeric and monomeric fractions demonstrates that both peaks contain tDGC.</p

Temperature-dependent unfolding of tDGC and of tDGC single mutants recorded by CD spectropolarimetry.

<p>The mean residue molar ellipticity vs temperature for proteins tDGC (dimeric form), tDGC (monomeric form, following treatment with RocR), D177A, E196A and R233A is plotted. A significant difference in the melting profiles of the wild type and the single tDGC mutants (disrupting individual salt bridge mutants) is visible. The melting temperature T_m, was determined by a Boltzmann sigmoid analysis (see text).</p

Refinement statistics.

<p>Refinement statistics.</p

Insight into Enzymatic Nitrile Reduction: QM/MM Study of the Catalytic Mechanism of QueF Nitrile Reductase

The NADPH-dependent QueF nitrile reductases catalyze the unprecedented four-electron reduction of nitrile to amine. QueF nitrile reductases can be found in the tRNA biosynthetic pathway of many bacteria and are potential antimicrobial drug targets. QueF enzymes have also attracted great attention as potential industrial biocatalysts for replacing the nitrile-reducing metal hydride catalysts used commonly in the chemical and pharmaceutical industries. Because of their narrow substrate specificity, engineering of the QueF enzymes to generate variants with altered or broadened substrate specificity is crucial for producing practically useful biocatalysts. A better understanding of the catalytic mechanism of the QueF enzymes would expedite rational inhibitor design and enzyme engineering. In this work, we probed the catalytic mechanism of the Vibrio cholerae QueF nitrile reductase by state of the art QM/MM calculations at the ONIOM­(B3LYP/6-311+G­(2d,2p):AMBER) level. The QM/MM computational results suggest that the nitrile to amine conversion proceeds through four major stages: (a) formation of a C–S covalent bond between the substrate and the catalytic cysteine residue to form the thioimidate intermediate, (b) hydride transfer from NADPH to the substrate to generate the thiohemiaminal intermediate, (c) cleavage of the C–S covalent bond to generate the imine intermediate, and (d) second hydride transfer from NADPH to the imine intermediate to generate the final amine product. The free energy barrier for the rate-limiting step, i.e. the second hydride transfer, was found to be 20.8 kcal/mol. The calculated barrier height and the catalytic residues identified as essential for nitrile reduction are in accordance with the currently available experimental data. The knowledge about the transition states, intermediates, and protein conformational changes along the reaction path will be valuable for the design of enzyme inhibitors as well as the engineering of QueF nitrile reductases

Synthesis of (<i>R</i>)-Mellein by a Partially Reducing Iterative Polyketide Synthase

Mellein and the related 3,4-dihydroisocoumarins are a family of natural products with interesting biological properties. The mechanisms of dihydroisocoumarin biosynthesis remain largely speculative today. Here we report the synthesis of mellein by a partially reducing iterative polyketide synthase (PR-PKS) as a pentaketide product. Remarkably, despite the head-to-tail homology shared with several fungal and bacterial PR-PKSs, the mellein synthase exhibits a distinct keto reduction pattern in the synthesis of the pentaketide. We present evidence to show that the ketoreductase (KR) domain alone is able to recognize and differentiate the polyketide intermediates, which provides a mechanistic explanation for the programmed keto reduction in these PR-PKSs