Dataset Information

Swit_4259, an acetoacetate decarboxylase-like enzyme from Sphingomonas wittichii RW1.

ABSTRACT: The Gram-negative bacterium Sphingomonas wittichii RW1 is notable for its ability to metabolize a variety of aromatic hydrocarbons. Not surprisingly, the S. wittichii genome contains a number of putative aromatic hydrocarbon-degrading gene clusters. One of these includes an enzyme of unknown function, Swit_4259, which belongs to the acetoacetate decarboxylase-like superfamily (ADCSF). Here, it is reported that Swit_4259 is a small (28.8?kDa) tetrameric ADCSF enzyme that, unlike the prototypical members of the superfamily, does not have acetoacetate decarboxylase activity. Structural characterization shows that the tertiary structure of Swit_4259 is nearly identical to that of the true decarboxylases, but there are important differences in the fine structure of the Swit_4259 active site that lead to a divergence in function. In addition, it is shown that while it is a poor substrate, Swit_4259 can catalyze the hydration of 2-oxo-hex-3-enedioate to yield 2-oxo-4-hydroxyhexanedioate. It is also demonstrated that Swit_4259 has pyruvate aldolase-dehydratase activity, a feature that is common to all of the family V ADCSF enzymes studied to date. The enzymatic activity, together with the genomic context, suggests that Swit_4259 may be a hydratase with a role in the metabolism of an as-yet-unknown hydrocarbon. These data have implications for engineering bioremediation pathways to degrade specific pollutants, as well as structure-function relationships within the ADCSF in general.

SUBMITTER: Mydy LS

PROVIDER: S-EPMC5713672 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Swit_4259, an acetoacetate decarboxylase-like enzyme from Sphingomonas wittichii RW1.

Mydy Lisa S LS Mashhadi Zahra Z Knight T William TW Fenske Tyler T Hagemann Trevor T Hoppe Robert W RW Han Lanlan L Miller Todd R TR Schwabacher Alan W AW Silvaggi Nicholas R NR

Acta crystallographica. Section F, Structural biology communications 20171114 Pt 12

The Gram-negative bacterium Sphingomonas wittichii RW1 is notable for its ability to metabolize a variety of aromatic hydrocarbons. Not surprisingly, the S. wittichii genome contains a number of putative aromatic hydrocarbon-degrading gene clusters. One of these includes an enzyme of unknown function, Swit_4259, which belongs to the acetoacetate decarboxylase-like superfamily (ADCSF). Here, it is reported that Swit_4259 is a small (28.8 kDa) tetrameric ADCSF enzyme that, unlike the prototypical ...[more]

PMID: 29199988

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Project description:Chlorinated congeners of dibenzo-p-dioxin and dibenzofuran are widely dispersed pollutants that can be treated using microorganisms, such as the Sphingomonas wittichii RW1 bacterium, able to transform some of them into non-toxic substances. The enzymes of the upper pathway for dibenzo-p-dioxin degradation in S. wittichii RW1 have been biochemically and genetically characterized, but its genome sequence has indicated the existence of a tremendous potential for aromatic compound transformation, with 56 ring-hydroxylating dioxygenase subunits, 34 extradiol dioxygenases, and 40 hydrolases. To further characterize this enzymatic arsenal, new methodological approaches should be employed. Here, a large shotgun proteomic survey has been performed on cells grown on dibenzofuran, dibenzo-p-dioxin, and 2-chlorodibenzo-p-dioxin, and compared to growth on acetate. Changes in the proteome were monitored over time. Peak lists were generated with the Mascot Daemon software (version 2.3.2; Matrix Science, London, UK) using the extract_msn.exe data import filter (Thermo Fisher Scientific) from the Xcalibur FT package (version 2.0.7; Thermo Fisher Scientific). Data import filter options were set to 400 (minimum mass), 5000 (maximum mass), 0 (grouping tolerance), 0 (intermediate scans) and 1000 (threshold). The mgf files from technical triplicates were merged, and MS/MS spectra were assigned using the Mascot Daemon 2.3.2 (Matrix Science) and a database containing the nonredundant RefSeq protein entries for S. wittichii RW1 (NCBI Taxonomy ID: 392499), comprising 5345 protein sequences totalling 1 800 684 amino acids. The search was performed using the following criteria: tryptic peptides with a maximum of two miscleavages, mass tolerances of 5 ppm on the parent ion and 0.5 Da on the MS/MS, fixed modification for carbamidomethylated cysteine, and variable modification for methionine oxidation. Mascot results were parsed using the IRMa 1.28.0 software. Peptides were identified with a P-value threshold below 0.05. Proteins were considered validated when at least two distinct peptides were detected. Using a selection of 11 result files and the appropriate decoy database, the false discovery rate for protein identification was estimated to be 0.33% with these parameters.

Project description:Sphingomonas wittichii RW1 is a bacterium isolated for its ability to degrade the xenobiotic compounds dibenzodioxin and dibenzofuran (DBF). A number of genes involved in DBF degradation have been previously characterized, such as the dxn cluster, dbfB, and the electron transfer components fdx1, fdx3, and redA2. Here we use a combination of whole genome transcriptome analysis and transposon library screening to characterize RW1 catabolic and other genes implicated in the reaction to or degradation of DBF. To detect differentially expressed genes upon exposure to DBF, we applied three different growth exposure experiments, using either short DBF exposures to actively growing cells or growing them with DBF as sole carbon and energy source. Genome-wide gene expression was examined using a custom-made microarray. In addition, proportional abundance determination of transposon insertions in RW1 libraries grown on salicylate or DBF by ultra-high throughput sequencing was used to infer genes whose interruption caused a fitness loss for growth on DBF. Expression patterns showed that batch and chemostat growth conditions, and short or long exposure of cells to DBF produced very different responses. Numerous other uncharacterized catabolic gene clusters putatively involved in aromatic compound metabolism increased expression in response to DBF. In addition, only very few transposon insertions completely abolished growth on DBF. Some of those (e.g., in dxnA1) were expected, whereas others (in a gene cluster for phenylacetate degradation) were not. Both transcriptomic data and transposon screening suggest operation of multiple redundant and parallel aromatic pathways, depending on DBF exposure. In addition, increased expression of other non-catabolic genes suggests that during initial exposure, S. wittichii RW1 perceives DBF as a stressor, whereas after longer exposure, the compound is recognized as a carbon source and metabolized using several pathways in parallel.

Project description:Sphingomonas wittichii strain RW1 (RW1) is one of the few strains that can grow on dibenzo-p-dioxin (DD). We conducted a transcriptomic study of RW1 using RNA-Seq to outline transcriptional responses to DD, dibenzofuran (DF), and the smectite clay mineral saponite with succinate as carbon source. The ability to grow on DD is rare compared to growth on the chemically similar DF even though the same initial dioxygenase may be involved in oxidation of both substrates. Therefore, we hypothesized the reason for this lies beyond catabolic pathways and may concern genes involved in processes for cell-substrate interactions such as substrate recognition, transport, and detoxification. Compared to succinate (SUC) as control carbon source, DF caused over 240 protein-coding genes to be differentially expressed, whereas more than 300 were differentially expressed with DD. Stress response genes were up-regulated in response to both DD and DF. This effect was stronger with DD than DF, suggesting a higher toxicity of DD compared to DF. Both DD and DF caused changes in expression of genes involved in active cross-membrane transport such as TonB-dependent receptor proteins, but the patterns of change differed between the two substrates. Multiple transcription factor genes also displayed expression patterns distinct to DD and DF growth. DD and DF induced the catechol ortho- and the salicylate/gentisate pathways, respectively. Both DD and DF induced the shared down-stream aliphatic intermediate compound pathway. Clay caused category-wide down-regulation of genes for cell motility and chemotaxis, particularly those involved in the synthesis, assembly and functioning of flagella. This is an environmentally important finding because clay is a major component of soil microbes' microenvironment influencing local chemistry and may serve as a geosorbent for toxic pollutants. Similar to clay, DD and DF also affected expression of genes involved in motility and chemotaxis.

Dataset Information

Swit_4259, an acetoacetate decarboxylase-like enzyme from Sphingomonas wittichii RW1.

Publications

Swit_4259, an acetoacetate decarboxylase-like enzyme from Sphingomonas wittichii RW1.

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