6 research outputs found

    Structural Evidence of a Productive Active Site Architecture for an Evolved Quorum-Quenching GKL Lactonase

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    The in vitro evolution and engineering of quorum-quenching lactonases with enhanced reactivities was achieved using a thermostable GKL enzyme as a template, yielding the E101G/R230C GKL mutant with increased catalytic activity and a broadened substrate range [Chow, J. Y., Xue, B., Lee, K. H., Tung, A., Wu, L., Robinson, R. C., and Yew, W. S. (2010) <i>J. Biol. Chem. 285</i>, 40911–40920]. This enzyme possesses the (β/α)<sub>8</sub>-barrel fold and is a member of the PLL (phosphotriesterase-like lactonase) group of enzymes within the amidohydrolase superfamily that hydrolyze <i>N</i>-acyl-homoserine lactones, which mediate the quorum-sensing pathways of bacteria. The structure of the evolved <i>N</i>-butyryl-l-homoserine lactone (substrate)-bound E101G/R230C GKL enzyme was determined, in the presence of the inactivating D266N mutation, to a resolution of 2.2 Å to provide an explanation for the observed rate enhancements. In addition, the substrate-bound structure of the catalytically inactive E101N/D266N mutant of the manganese-reconstituted enzyme was determinied to a resolution of 2.1 Å and the structure of the ligand-free, manganese-reconstituted E101N mutant to a resolution of 2.6 Å, and the structures of ligand-free zinc-reconstituted wild-type, E101N, R230D, and E101G/R230C mutants of GKL were determinied to resolutions of 2.1, 2.1, 1.9, and 2.0 Å, respectively. In particular, the structure of the evolved E101G/R230C mutant of GKL provides evidence of a catalytically productive active site architecture that contributes to the observed enhancement of catalysis. At high concentrations, wild-type and mutant GKL enzymes are differentially colored, with absorbance maxima in the range of 512–553 nm. The structures of the wild-type and mutant GKL provide a tractable link between the origins of the coloration and the charge-transfer complex between the α-cation and Tyr99 within the enzyme active site. Taken together, this study provides evidence of the modulability of enzymatic catalysis through subtle changes in enzyme active site architecture

    Discovery of <i>cis</i>-regulatory motifs.

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    <p>Motifs were identified by analysing upstream sequences of constituent genes or operons in each cluster. The asterisk (*) indicates that the motif was detected using MEME and BioProspector. Tick symbols indicate that all cluster genes have a cognate homolog in the specified species (i.e. 100%), otherwise the proportion of homologs in that species is reported. Filled circles indicate that the discovered <i>cis</i>-motifs in Bp are significantly similar () to Bt or Bm. Motifs that match to known binding sites and corresponding binding proteins in other species are reported in the last column. Bt, <i>B. thailandensis</i>; Bm, <i>B. mallei</i>.</p

    Expressed transcripts in the Bp condition compendium.

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    <p>High-resolution views of different genomic features are depicted. All transcripts depicted were expressed above the median cut-off threshold. (A) Transcriptional annotation of the <i>Burkholderia pseudomallei</i> K96243 reference genome. The transcriptome map is presented along the chromosomal coordinates in a strand-specific manner, with the outermost track composed of Sanger annotated genes (orange), followed by novel genes (green), the Bp operons (purple) and finally the non-coding RNAs (ncRNAs; red). In all tracks, predicted genomic features that do not have an associated transcript in this study are colored in grey. The genes, operons and ncRNAs are arranged in a strand-specific manner by visualizing them in either the forward (+) or the reverse (−) tracks. The black vertical lines indicate the start/stop sites of the circular chromosomes. (B) Sanger genes and novel genes. Expressed strand-specific transcripts are presented as blue bars along the forward and reverse strands. Transcript boundaries correspond to predicted start and stop coordinates of Sanger annotated genes and FGENESB novel genes. (C) Differential expression of a Bp operon. Expression of a predicted flagella operon (<i>BPSL0026 – BPSL0032</i>) in a specific condition (taurine exposure). (D) Antisense transcription. <i>BPSL0095</i>, a gene coding for hypothetical protein exhibits antisense transcription upon exposure to human serum.</p

    Bp chromosomes display distinct transcriptional landscapes.

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    <p>(A) Cumulative curves for expression of genes across the condition compendium. The graph represents the percentage of new genes expressed on Chr 1 (red) and Chr 2 (green) (y-axis) upon the successive addition of conditions (x-axis). This analysis was confined to Sanger genes to minimize annotation errors. (B) Chr 1 and Chr 2 exhibit constitutive and mosaic expression respectively. The graph relates the proportion of genes expressed on each chromosome (y-axis) under any particular number of conditions (x-axis). Chr 1 genes are expressed in most conditions (rightward upslope, red), while Chr 2 genes are expressed in specific conditions (leftward upslope, green). (C) Chr 1 genes exhibit higher expression levels than Chr 2 genes. Each dot represents the median expression of all detectably expressed genes on the respective chromosome, joined by the same condition. Chromosomal expression levels were compared using one-tailed paired t-test ().</p

    Co-expression network of Bp condition-dependent transcription.

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    <p>(A) Co-expression network. Nodes are individual genes, connected to one another by significant co-expression relationships (mutual information score ). The colours represent clusters over-represented in different Riley annotations, and their respective annotations are provided at the bottom. (B) Condition dependent cluster expression. The heat-map depicts representative clusters and patterns of expression across conditions. Gene expression levels were mean-normalized. (C) Inter-cluster relationships. The MRCN unit M036 consists of two clusters: C131 and C265, which include genes encoding proteins for degrading misfolded proteins and other genes with hypothetical functions. Thickness of edges represents the strength of the co-expression relationship between two genes. (D) Condition groups. The different condition-specific transcriptional profiles were clustered to one another based on similarities in expression of genes from the Bp core genome. Condition groups deemed to be stable by bootstrap assessment are marked in colors.</p

    Identification of Bp ncRNAs.

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    <p>(A) Condition-dependence of ncRNA expression. The heat-map depicts 766 identified ncRNAs and their patterns of expression across the condition compendium. Red depicts high expression, and green depicts low expression. (B) <i>BPNC10061R</i> expression is triggered by sorbitol. <i>BPNC10061R</i> is highly expressed under condition of osmotic stress (2M Sorbitol) compared to desiccation. (C) Secondary structure and species conservation of <i>BPNC10061R</i>. Consensus sequences homologous to <i>BPNC10061R</i> are found in <i>B. mallei</i>, <i>B. cenocepacia</i> and <i>B. thailandensis</i> strains. The sequences were aligned, and corresponding secondary structures were predicted.</p