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Crystallization of purple nitrous oxide reductase from Pseudomonas stutzeri.

ABSTRACT: Nitrous oxide reductase (N(2)OR) from Pseudomonas stutzeri catalyzes the final step in denitrification: the two-electron reduction of nitrous oxide to molecular dinitrogen. Crystals of the enzyme were grown under strict exclusion of dioxygen by sitting-drop vapour diffusion using 2R,3R-butanediol as a cryoprotectant. N(2)OR crystallized in either space group P1 or P6(5). Interestingly, the key determinant for the resulting space group was the crystallization temperature. Crystals belonging to space group P1 contained four 130?kDa dimers in the asymmetric unit, while crystals belonging to space group P6(5) contained a single dimer in the asymmetric unit. Diffraction data were collected to resolutions better than 2?Å.

SUBMITTER: Pomowski A

PROVIDER: S-EPMC3001670 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Crystallization of purple nitrous oxide reductase from Pseudomonas stutzeri.

Pomowski Anja A Zumft Walter G WG Kroneck Peter M H PM Einsle Oliver O

Acta crystallographica. Section F, Structural biology and crystallization communications 20101029 Pt 11

Nitrous oxide reductase (N(2)OR) from Pseudomonas stutzeri catalyzes the final step in denitrification: the two-electron reduction of nitrous oxide to molecular dinitrogen. Crystals of the enzyme were grown under strict exclusion of dioxygen by sitting-drop vapour diffusion using 2R,3R-butanediol as a cryoprotectant. N(2)OR crystallized in either space group P1 or P6(5). Interestingly, the key determinant for the resulting space group was the crystallization temperature. Crystals belonging to sp ...[more]

PMID: 21045317

Similar Datasets

Role of the Tat ransport system in nitrous oxide reductase translocation and cytochrome cd1 biosynthesis in Pseudomonas stutzeri.

Project description:By transforming N2O to N2, the multicopper enzyme nitrous oxide reductase provides a periplasmic electron sink for a respiratory chain that is part of denitrification. The signal sequence of the enzyme carries the heptameric twin-arginine consensus motif characteristic of the Tat pathway. We have identified tat genes of Pseudomonas stutzeri and functionally analyzed the unlinked tatC and tatE loci. A tatC mutant retained N2O reductase in the cytoplasm in the unprocessed form and lacking the metal cofactors. This is contrary to viewing the Tat system as specific only for fully assembled proteins. A C618V exchange in the electron transfer center CuA rendered the enzyme largely incompetent for transport. The location of the mutation in the C-terminal domain of N(2)O reductase implies that the Tat system acts on a completely synthesized protein and is sensitive to a late structural variation in folding. By generating a tatE mutant and a reductase-overproducing strain, we show a function for TatE in N2O reductase translocation. Further, we have found that the Tat and Sec pathways have to cooperate to produce a functional nitrite reductase system. The cytochrome cd1 nitrite reductase was found in the periplasm of the tatC mutant, suggesting export by the Sec pathway; however, the enzyme lacked the heme D1 macrocycle. The NirD protein as part of a complex required for heme D1 synthesis or processing carries a putative Tat signal peptide. Since NO reduction was also inhibited in the tatC mutant, the Tat protein translocation system is necessary in multiple ways for establishing anaerobic nitrite denitrification.

| S-EPMC95051 | biostudies-literature

NosR, a membrane-bound regulatory component necessary for expression of nitrous oxide reductase in denitrifying Pseudomonas stutzeri.

Project description:The regulatory element NosR was identified within the nos region of the denitrification gene cluster of Pseudomonas stutzeri ZoBell (ATCC 14405) and characterized. It is essential for expression of the N2O reductase encoded by nosZ immediately downstream of nosR. The nosR region was initially identified by Tn5 mutagenesis (W. G. Zumft, K. Döhler, and H. Körner, J. Bacteriol. 163:918-924, 1985). It consists of a single open reading frame of 2,172 nucleotides and has the coding capacity for an 81.9-kDa protein. The codon usage for nosR, with its high G + C content of 62.4 mol% and a preference for G or C at the third position, is characteristic for a Pseudomonas gene. Hydropathy analysis classified NosR as an integral membrane protein with at least seven membrane-spanning segments. No similarity to known bacterial regulator proteins was found in a data bank search. However, the C terminus of NosR shows sequence similarity to the cysteine clusters of several 2[4Fe-4S] bacterial ferrodoxins. A monocistronic mRNA for nosZ which allowed us to monitor NosR function was identified. Complementation of Nos- mutant MK418 (nosR::Tn5) with the nosR gene supplied in trans restored nosZ transcription and expression of a catalytically active N2O reductase. In addition to evidence of the requirement for NosR, indirect evidence for involvement of the transcriptional regulator Fnr is presented.

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Potential of aerobic denitrification by Pseudomonas stutzeri TR2 to reduce nitrous oxide emissions from wastewater treatment plants.

Project description:In contrast to most denitrifiers studied so far, Pseudomonas stutzeri TR2 produces low levels of nitrous oxide (N(2)O) even under aerobic conditions. We compared the denitrification activity of strain TR2 with those of various denitrifiers in an artificial medium that was derived from piggery wastewater. Strain TR2 exhibited strong denitrification activity and produced little N(2)O under all conditions tested. Its growth rate under denitrifying conditions was near comparable to that under aerobic conditions, showing a sharp contrast to the lower growth rates of other denitrifiers under denitrifying conditions. Strain TR2 was tolerant to toxic nitrite, even utilizing it as a good denitrification substrate. When both nitrite and N(2)O were present, strain TR2 reduced N(2)O in preference to nitrite as the denitrification substrate. This bacterial strain was readily able to adapt to denitrifying conditions by expressing the denitrification genes for cytochrome cd(1) nitrite reductase (NiR) (nirS) and nitrous oxide reductase (NoS) (nosZ). Interestingly, nosZ was constitutively expressed even under nondenitrifying, aerobic conditions, consistent with our finding that strain TR2 preferred N(2)O to nitrite. These properties of strain TR2 concerning denitrification are in sharp contrast to those of well-characterized denitrifiers. These results demonstrate that some bacterial species, such as strain TR2, have adopted a strategy for survival by preferring denitrification to oxygen respiration. The bacterium was also shown to contain the potential to reduce N(2)O emissions when applied to sewage disposal fields.

| S-EPMC2901746 | biostudies-literature

Experimental evidence for a link among cupredoxins: red, blue, and purple copper transformations in nitrous oxide reductase.

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| S-EPMC2430339 | biostudies-literature

Operon structure and regulation of the nos gene region of Pseudomonas stutzeri, encoding an ABC-Type ATPase for maturation of nitrous oxide reductase.

Project description:The synthesis of a functional nitrous oxide reductase requires an assembly apparatus for the insertion of the prosthetic copper. Part of the system is encoded by maturation genes located in Pseudomonas stutzeri immediately downstream of the structural gene for the enzyme. We have studied the transcriptional organization and regulation of this region and found a nosDFYL tatE operon structure. In addition to a putative ABC transporter, consisting of NosD, NosF, and NosY, the operon encodes a Cu chaperone, NosL, and a component of the Tat translocon, TatE. The nosD operon was activated in response to anaerobiosis and nitrate denitrification. The membrane-bound regulator NosR was required for operon expression; in addition, DnrD, a regulator of the Crp-Fnr family, enhanced expression under anaerobic conditions. This establishes a likely signal transduction sequence of NO --> DnrD --> nosR/NosR --> nosD operon. DnrD-dependent expression was also observed for the nnrS operon (located immediately downstream of the nosD operon), which encodes a putative heme-Cu protein (NnrS) and a member of the short-chain dehydrogenase family (ORF247). The NosF protein, encoded within the nosD operon, exhibits sequence similarity to ABC-type ATPases. It was fused to the Escherichia coli maltose-binding protein and overexpressed in soluble form. The fusion protein was purified and shown to have ATPase activity. NosF is the first maturation factor for which a catalytic function has been demonstrated in vitro.

| S-EPMC150149 | biostudies-other

A decade of crystallization drops: crystallization of the cbb3 cytochrome c oxidase from Pseudomonas stutzeri.

Project description:The cbb3 cytochrome c oxidases are distant members of the superfamily of heme copper oxidases. These terminal oxidases couple O2 reduction with proton transport across the plasma membrane and, as a part of the respiratory chain, contribute to the generation of an electrochemical proton gradient. Compared with other structurally characterized members of the heme copper oxidases, the recently determined cbb3 oxidase structure at 3.2 Å resolution revealed significant differences in the electron supply system, the proton conducting pathways and the coupling of O2 reduction to proton translocation. In this paper, we present a detailed report on the key steps for structure determination. Improvement of the protein quality was achieved by optimization of the number of lipids attached to the protein as well as the separation of two cbb3 oxidase isoenzymes. The exchange of n-dodecyl-?-D-maltoside for a precisely defined mixture of two ?-maltosides and decanoylsucrose as well as the choice of the crystallization method had a most profound impact on crystal quality. This report highlights problems frequently encountered in membrane protein crystallization and offers meaningful approaches to improve crystal quality.

| S-EPMC3970892 | biostudies-literature

Unexpected nondenitrifier nitrous oxide reductase gene diversity and abundance in soils.

Project description:Agricultural and industrial practices more than doubled the intrinsic rate of terrestrial N fixation over the past century with drastic consequences, including increased atmospheric nitrous oxide (N(2)O) concentrations. N(2)O is a potent greenhouse gas and contributor to ozone layer destruction, and its release from fixed N is almost entirely controlled by microbial activities. Mitigation of N(2)O emissions to the atmosphere has been attributed exclusively to denitrifiers possessing NosZ, the enzyme system catalyzing N(2)O to N(2) reduction. We demonstrate that diverse microbial taxa possess divergent nos clusters with genes that are related yet evolutionarily distinct from the typical nos genes of denitirifers. nos clusters with atypical nosZ occur in Bacteria and Archaea that denitrify (44% of genomes), do not possess other denitrification genes (56%), or perform dissimilatory nitrate reduction to ammonium (DNRA; (31%). Experiments with the DNRA soil bacterium Anaeromyxobacter dehalogenans demonstrated that the atypical NosZ is an effective N(2)O reductase, and PCR-based surveys suggested that atypical nosZ are abundant in terrestrial environments. Bioinformatic analyses revealed that atypical nos clusters possess distinctive regulatory and functional components (e.g., Sec vs. Tat secretion pathway in typical nos), and that previous nosZ-targeted PCR primers do not capture the atypical nosZ diversity. Collectively, our results suggest that nondenitrifying populations with a broad range of metabolisms and habitats are potentially significant contributors to N(2)O consumption. Apparently, a large, previously unrecognized group of environmental nosZ has not been accounted for, and characterizing their contributions to N(2)O consumption will advance understanding of the ecological controls on N(2)O emissions and lead to refined greenhouse gas flux models.

| S-EPMC3511753 | biostudies-literature

Diversity of nitrous oxide reductase (nosZ) genes in continental shelf sediments.

Project description:Diversity of the nitrous oxide reductase (nosZ) gene was examined in sediments obtained from the Atlantic Ocean and Pacific Ocean continental shelves. Approximately 1,100 bp of the nosZ gene were amplified via PCR, using nosZ gene-specific primers. Thirty-seven unique copies of the nosZ gene from these marine environments were characterized, increasing the nosZ sequence database fourfold. The average DNA similarity for comparisons between all 49 variants of the nosZ gene was 64% +/- 10%. Alignment of the derived amino acid sequences confirmed the conservation of important structural motifs. A highly conserved region is proposed as the copper binding, catalytic site (CuZ) of the mature protein. Phylogenetic analysis demonstrated three major clusters of nosZ genes, with little overlap between environmental and culture-based groups. Finally, the two non-culture-based gene clusters generally corresponded to sampling location, implying that denitrifier communities may be restricted geographically.

| S-EPMC91238 | biostudies-literature

Functional assembly of nitrous oxide reductase provides insights into copper site maturation.

Project description:The multicopper enzyme nitrous oxide reductase reduces the greenhouse gas N2O to uncritical N2 as the final step of bacterial denitrification. Its two metal centers require an elaborate assembly machinery that so far has precluded heterologous production as a prerequisite for bioremediatory applications in agriculture and wastewater treatment. Here, we report on the production of active holoenzyme in Escherichia coli using a two-plasmid system to produce the entire biosynthetic machinery as well as the structural gene for the enzyme. Using this recombinant system to probe the role of individual maturation factors, we find that the ABC transporter NosFY and the accessory NosD protein are essential for the formation of the [4Cu:2S] site CuZ, but not the electron transfer site CuA Depending on source organism, the heterologous host E. coli can, in some cases, compensate for the lack of the Cu chaperone NosL, while in others this protein is strictly required, underlining the case for designing a recombinant system to be entirely self-contained.

| S-EPMC6601269 | biostudies-literature

Crystallization and preliminary X-ray diffraction studies of L-rhamnose isomerase from Pseudomonas stutzeri.

Project description:L-Rhamnose isomerase from Pseudomonas stutzeri (P. stutzeri L-RhI) catalyzes not only the reversible isomerization of L-rhamnose to L-rhamnulose, but also isomerization between various rare aldoses and ketoses. Purified His-tagged P. stutzeri L-RhI was crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 74.3, b = 104.0, c = 107.0 A, beta = 106.8 degrees . Diffraction data have been collected to 2.0 A resolution. The molecular weight of the purified P. stutzeri L-RhI with a His tag at the C-terminus was confirmed to be 47.7 kDa by MALDI-TOF mass-spectrometric analysis and the asymmetric unit is expected to contain four molecules.

| S-EPMC2243077 | biostudies-literature

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