The nature of the copper centres in nitrous oxide reductase (Pseudomonas stutzeri)

ABSTRACT 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.

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Expression of the nos operon proteins from Pseudomonas stutzeri in transgenic plants to assemble nitrous oxide reductase

Transgenic Research ◽

10.1007/s11248-011-9555-1 ◽

2011 ◽

Vol 21 (3) ◽

pp. 593-603 ◽

Cited By ~ 7

Author(s):

Shen Wan ◽

Yaseen Mottiar ◽

Amanda M. Johnson ◽

Kagami Goto ◽

Illimar Altosaar

Keyword(s):

Nitrous Oxide ◽

Transgenic Plants ◽

Pseudomonas Stutzeri ◽

Nitrous Oxide Reductase

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nosX is essential for whole-cell N2O reduction in Paracoccus denitrificans but not for assembly of copper centres of nitrous oxide reductase

Microbiology ◽

10.1099/mic.0.000955 ◽

2020 ◽

Vol 166 (10) ◽

pp. 909-917 ◽

Cited By ~ 1

Author(s):

Sophie P. Bennett ◽

Maria J. Torres ◽

Manuel J. Soriano-Laguna ◽

David J. Richardson ◽

Andrew J. Gates ◽

...

Keyword(s):

Nitrous Oxide ◽

Type Species ◽

Paracoccus Denitrificans ◽

Pseudomonas Stutzeri ◽

Reduction Reaction ◽

Nitrous Oxide Reductase ◽

Content Type ◽

Link Type ◽

Reverse Transcription Pcr

Nitrous oxide (N2O) is a potent greenhouse gas that is produced naturally as an intermediate during the process of denitrification carried out by some soil bacteria. It is consumed by nitrous oxide reductase (N2OR), the terminal enzyme of the denitrification pathway, which catalyses a reduction reaction to generate dinitrogen. N2OR contains two important copper cofactors (CuA and CuZ centres) that are essential for activity, and in copper-limited environments, N2OR fails to function, contributing to rising levels of atmospheric N2O and a major environmental challenge. Here we report studies of nosX, one of eight genes in the nos cluster of the soil dwelling α-proteobaterium Paraccocus denitrificans. A P. denitrificans ΔnosX deletion mutant failed to reduce N2O under both copper-sufficient and copper-limited conditions, demonstrating that NosX plays an essential role in N2OR activity. N2OR isolated from nosX-deficient cells was found to be unaffected in terms of the assembly of its copper cofactors, and to be active in in vitro assays, indicating that NosX is not required for the maturation of the enzyme; in particular, it plays no part in the assembly of either of the CuA and CuZ centres. Furthermore, quantitative Reverse Transcription PCR (qRT-PCR) studies showed that NosX does not significantly affect the expression of the N2OR-encoding nosZ gene. NosX is a homologue of the FAD-binding protein ApbE from Pseudomonas stutzeri , which functions in the flavinylation of another N2OR accessory protein, NosR. Thus, it is likely that NosX is a system-specific maturation factor of NosR, and so is indirectly involved in maintaining the reaction cycle of N2OR and cellular N2O reduction.

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A novel nitrous oxide mitigation strategy: expressing nitrous oxide reductase fromPseudomonas stutzeriin transgenic plants

Canadian Journal of Plant Science ◽

10.4141/cjps2013-141 ◽

2014 ◽

Vol 94 (6) ◽

pp. 1013-1023 ◽

Cited By ~ 2

Author(s):

Shen Wan ◽

Trevor Greenham ◽

Kagami Goto ◽

Yaseen Mottiar ◽

Amanda M. Johnson ◽

...

Keyword(s):

Nitrous Oxide ◽

Transgenic Plants ◽

Stratospheric Ozone ◽

Pseudomonas Stutzeri ◽

Mitigation Strategy ◽

Nitrous Oxide Reductase ◽

Adaptation To Climate Change ◽

Tobacco Plants ◽

Nosz Gene ◽

Nitrous Oxide Mitigation

Wan, S., Greenham, T., Goto, K., Mottiar, Y., Johnson, A. M., Staebler, J. M., Zaidi, M. A., Shu, Q. and Altosaar, I. 2014. A novel nitrous oxide mitigation strategy: expressing nitrous oxide reductase from Pseudomonas stutzeri in transgenic plants. Can. J. Plant Sci. 94: 1013–1025. As a stable greenhouse gas, nitrous oxide (N2O) plays a significant role in stratospheric ozone destruction. The primary anthropogenic N2O source is the use of nitrogen in agriculture. Currently, the annual N2O emissions from this soil–plant–microbial system is more than 2.6 Tg (1 Tg=1 million metric tonnes) of N2O-N globally. So it is important to explore some innovative and effective biology-based strategies for N2O mitigation. If shown to be effective in field trails as well as laboratory-scale experiments, such GMO plants could help guide international policies on adaptation to climate change. The bacterial enzyme nitrous oxide reductase (N2OR) is the only known enzyme capable of catalyzing the final step of the denitrification pathway, conversion of N2O to N2. To “scrub” the N2O emissions, bacterial N2OR was heterologously expressed in plants. Structurally, the enzyme N2OR is encoded by nosZ, but its biosynthesis and assembly in prokaryotes require the products of several nos genes, including a putative ABC-type transporter encoded by nosDFY, and the copper chaperone NosL for biogenesis of the metal centre. We have generated transgenic tobacco plants expressing the nosZ gene, as well as tobacco plants in which the other nos genes were co-expressed under the control of a root-specific promoter (rolD) and a constitutive promoter (d35S). The nosZ gene from Pseudomonas stutzeri heterologously expressed in tobacco produced active recombinant N2OR. The positive results in the preliminary proof-of-principle experiments indicated that plants heterologously expressing N2OR could mitigate emissions at the source before N2O reaches the stratosphere or troposphere.

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

Journal of Bacteriology ◽

10.1128/jb.174.16.5332-5339.1992 ◽

1992 ◽

Vol 174 (16) ◽

pp. 5332-5339 ◽

Cited By ~ 51

Author(s):

H Cuypers ◽

A Viebrock-Sambale ◽

W G Zumft

Keyword(s):

Nitrous Oxide ◽

Pseudomonas Stutzeri ◽

Nitrous Oxide Reductase ◽

Membrane Bound

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On the nature of the CuA-site in nitrous oxide reductase from Pseudomonas stutzeri

Journal of Inorganic Biochemistry ◽

10.1016/0162-0134(95)97776-m ◽

1995 ◽

Vol 59 (2-3) ◽

pp. 688

Author(s):

F. Neese ◽

W.G. Zumft ◽

W.E. Antholine ◽

P.M.H. Kroneck

Keyword(s):

Nitrous Oxide ◽

Pseudomonas Stutzeri ◽

Nitrous Oxide Reductase

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Bacterial nitrous oxide reductase expressed in transgenic plants: Evidence for sufficient anaerobicity to permit activity

Canadian Journal of Plant Science ◽

10.4141/cjps2011-284 ◽

2012 ◽

Vol 92 (7) ◽

pp. 1283-1294

Author(s):

Shen Wan ◽

Kagami Goto ◽

Yaseen Mottiar ◽

Julianne M. Staebler ◽

Amanda M. Johnson ◽

...

Keyword(s):

Nitrous Oxide ◽

Transgenic Plants ◽

Crop Yields ◽

Pseudomonas Stutzeri ◽

Genetically Engineered ◽

Nitrous Oxide Reductase ◽

In Planta ◽

Accessory Gene ◽

Genetically Engineered Crops ◽

Anaerobic Environment

Wan, S., Goto, K., Mottiar, Y., Staebler, J. M., Johnson, A. M., Voronova, A., Blais, D. R., Zaidi, M. A. and Altosaar, I. 2012. Bacterial nitrous oxide reductase expressed in transgenic plants: Evidence for sufficient anaerobicity to permit activity. Can. J. Plant Sci. 92: 1283–1294. Soil nitrogen enrichment practices enhance crop yields but greatly exacerbate the production and release of nitrous oxide (N2O), a potent greenhouse gas. The conversion of N2O to dinitrogen (N2) in soil denitrifiers is normally driven by the anaerobic enzyme nitrous oxide reductase (N2OR). Since this step is often limited in fertilised soils, we have transferred this unique microbial biocatalyst from the soil bacterium Pseudomonas stutzeri to transgenic Nicotiana tabacum plants. Our results confirm that engineered plants expressing the N2OR structural gene nosZ are capable of producing functional reductase enzyme without the involvement of the native accessory gene products. Since bacterial N2OR normally exists in the anaerobic environment of the soil bacterium's periplasmic space, this novel in planta activity suggests that plant cells can provide a similar hypoxic refuge for anaerobic enzyme activity. Genetically engineered crops containing N2OR could have considerable potential in the phytoremediation of atmospheric N2O.

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