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Expression, purification, crystallization and preliminary X-ray analysis of para-nitrophenol 4-monooxygenase from Pseudomonas putida DLL-E4.

ABSTRACT: Para-nitrophenol 4-monooxygenase (PnpA) plays an important role in bacterial degradation of para-nitrophenol by oxidative release of the nitro group from the aromatic ring to form p-benzoquinone. In order to understand the structural basis of the function of this enzyme, PnpA was cloned, expressed in Escherichia coli and purified. PnpA was crystallized by the hanging-drop vapour-diffusion technique with PEG 4000 as precipitant. The PnpA crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 54.47, b = 77.56, c = 209.17 A, and diffracted to 2.24 A resolution.

SUBMITTER: Liu W

PROVIDER: S-EPMC2765886 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Expression, purification, crystallization and preliminary X-ray analysis of para-nitrophenol 4-monooxygenase from Pseudomonas putida DLL-E4.

Liu Weidong W Shen Wenjing W Zhao Xiaoli X Cao Hui H Cui Zhongli Z

Acta crystallographica. Section F, Structural biology and crystallization communications 20090923 Pt 10

Para-nitrophenol 4-monooxygenase (PnpA) plays an important role in bacterial degradation of para-nitrophenol by oxidative release of the nitro group from the aromatic ring to form p-benzoquinone. In order to understand the structural basis of the function of this enzyme, PnpA was cloned, expressed in Escherichia coli and purified. PnpA was crystallized by the hanging-drop vapour-diffusion technique with PEG 4000 as precipitant. The PnpA crystals belonged to space group P2(1)2(1)2(1), with unit-c ...[more]

PMID: 19851007

Similar Datasets

The Regulation of para-Nitrophenol Degradation in Pseudomonas putida DLL-E4.

Project description:Pseudomonas putida DLL-E4 can efficiently degrade para-nitrophenol and its intermediate metabolite hydroquinone. The regulation of para-nitrophenol degradation was studied, and PNP induced a global change in the transcriptome of P. putida DLL-E4. When grown on PNP, the wild-type strain exhibited significant downregulation of 2912 genes and upregulation of 845 genes, whereas 2927 genes were downregulated and 891 genes upregulated in a pnpR-deleted strain. Genes related to two non-coding RNAs (ins1 and ins2), para-nitrophenol metabolism, the tricarboxylic acid cycle, the outer membrane porin OprB, glucose dehydrogenase Gcd, and carbon catabolite repression were significantly upregulated when cells were grown on para-nitrophenol plus glucose. pnpA, pnpR, pnpC1C2DECX1X2, and pnpR1 are key genes in para-nitrophenol degradation, whereas pnpAb and pnpC1bC2bDbEbCbX1bX2b have lost the ability to degrade para-nitrophenol. Multiple components including transcriptional regulators and other unknown factors regulate para-nitrophenol degradation, and the transcriptional regulation of para-nitrophenol degradation is complex. Glucose utilization was enhanced at early stages of para-nitrophenol supplementation. However, it was inhibited after the total consumption of para-nitrophenol. The addition of glucose led to a significant enhancement in para-nitrophenol degradation and up-regulation in the expression of genes involved in para-nitrophenol degradation and carbon catabolite repression (CCR). It seemed that para-nitrophenol degradation can be regulated by CCR, and relief of CCR might contribute to enhanced para-nitrophenol degradation. In brief, the regulation of para-nitrophenol degradation seems to be controlled by multiple factors and requires further study.

| S-EPMC4871426 | biostudies-literature

Complete Genome Sequence of the p-Nitrophenol-Degrading Bacterium Pseudomonas putida DLL-E4.

Project description:The first complete genome sequence of a p-nitrophenol (PNP)-degrading bacterium is reported here. Pseudomonas putida DLL-E4, a Gram-negative bacterium isolated from methyl-parathion-polluted soil, can utilize PNP as the sole carbon and nitrogen source. P. putida DLL-E4 has a 6,484,062 bp circular chromosome that contains 5,894 genes, with a G+C content of 62.46%.

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Purification, crystallization and preliminary X-ray diffraction of human S100A15.

Project description:Human S100A15 is a novel member of the S100 family of EF-hand calcium-binding proteins and was recently identified in psoriasis, where it is significantly upregulated in lesional skin. The protein is implicated as an effector in calcium-mediated signal transduction pathways. Although its biological function is unclear, the association of the 11.2 kDa S100A15 with psoriasis suggests that it contributes to the pathogenesis of the disease and could provide a molecular target for therapy. To provide insight into the function of S100A15, the protein was crystallized to visualize its structure and to further the understanding of how the many similar calcium-binding mediator proteins in the cell distinguish their cognate target molecules. The S100A15 protein has been cloned, expressed and purified to homogeneity and produced two crystal forms. Crystals of form I are triclinic, with unit-cell parameters a = 33.5, b = 44.3, c = 44.8 angstroms, alpha = 71.2, beta = 68.1, gamma = 67.8 degrees and an estimated two molecules in the asymmetric unit, and diffract to 1.7 angstroms resolution. Crystals of form II are monoclinic, with unit-cell parameters a = 82.1, b = 33.6, c = 52.2 angstroms, beta = 128.2 degrees and an estimated one molecule in the asymmetric unit, and diffract to 2.0 angstroms resolution. This structural analysis of the human S100A15 will further aid in the phylogenic comparison between the other members of the S100 protein family, especially the highly homologous paralog S100A7.

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Pseudomonas putida strain:DLL-E4

Project description:Pseudomonas putida strain:DLL-E4 Genome sequencing

| PRJNA244741 | ENA

Pseudomonas putida strain:DLL-E4

Project description:E4-GP

| PRJNA283348 | ENA

Pseudomonas putida strain:DLL-E4

Project description:Transcriptome of Pseudomonas putida strain DLL-E4

| PRJNA283347 | ENA

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of Enpp6.

Project description:Enpp (ectonucleotide phosphodiesterase/pyrophosphatase) 6 is a membrane-bound glycoprotein that hydrolyzes choline-containing compounds such as lysophosphatidylcholine and glycerophosphorylcholine, and presumably participates in choline metabolism. The catalytic domain of mouse Enpp6 was expressed in HEK293T cells, purified using the TARGET tag/P20.1-Sepharose system and crystallized. An X-ray diffraction data set was collected to 1.8?Å resolution. The crystal belonged to space group P1, with unit-cell parameters a=63.7, b=68.8, c=69.7?Å, ?=60.6, ?=87.0, ?=68.1°. Assuming the presence of two protein molecules per asymmetric unit, the solvent content was estimated to be 49.5%.

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Expression, purification, crystallization and preliminary X-ray crystallographic analysis of Enpp1.

Project description:Enpp1 is an extracellular membrane-bound glycoprotein that regulates bone mineralization by hydrolyzing ATP to generate pyrophosphate. The extracellular region of mouse Enpp1 was expressed in HEK293S GnT1(-) cells, purified using the TARGET tag/P20.1-Sepharose system and crystallized. An X-ray diffraction data set was collected to 3.0?Å resolution. The crystal belonged to space group P3(1), with unit-cell parameters a = b = 105.3, c = 173.7?Å. A single-wavelength anomalous dispersion (SAD) data set was also collected to 2.7?Å resolution using a selenomethionine-labelled crystal. The experimental phases determined by the SAD method produced an interpretable electron-density map.

| S-EPMC3388920 | biostudies-literature

Purification, crystallization and preliminary X-ray diffraction analysis of human chondroadherin.

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| S-EPMC2496858 | biostudies-literature

A Two-Component para-Nitrophenol Monooxygenase Initiates a Novel 2-Chloro-4-Nitrophenol Catabolism Pathway in Rhodococcus imtechensis RKJ300.

Project description:Rhodococcus imtechensis RKJ300 (DSM 45091) grows on 2-chloro-4-nitrophenol (2C4NP) and para-nitrophenol (PNP) as the sole carbon and nitrogen sources. In this study, by genetic and biochemical analyses, a novel 2C4NP catabolic pathway different from those of all other 2C4NP utilizers was identified with hydroxyquinol (hydroxy-1,4-hydroquinone or 1,2,4-benzenetriol [BT]) as the ring cleavage substrate. Real-time quantitative PCR analysis indicated that the pnp cluster located in three operons is likely involved in the catabolism of both 2C4NP and PNP. The oxygenase component (PnpA1) and reductase component (PnpA2) of the two-component PNP monooxygenase were expressed and purified to homogeneity, respectively. The identification of chlorohydroquinone (CHQ) and BT during 2C4NP degradation catalyzed by PnpA1A2 indicated that PnpA1A2 catalyzes the sequential denitration and dechlorination of 2C4NP to BT and catalyzes the conversion of PNP to BT. Genetic analyses revealed that pnpA1 plays an essential role in both 2C4NP and PNP degradations by gene knockout and complementation. In addition to catalyzing the oxidation of CHQ to BT, PnpA1A2 was also found to be able to catalyze the hydroxylation of hydroquinone (HQ) to BT, revealing the probable fate of HQ that remains unclear in PNP catabolism by Gram-positive bacteria. This study fills a gap in our knowledge of the 2C4NP degradation mechanism in Gram-positive bacteria and also enhances our understanding of the genetic and biochemical diversity of 2C4NP catabolism.

| S-EPMC4711147 | biostudies-literature

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