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Grants from the EC |
Rob van Spanning Department of Molecular Cell Physiology vrije Universiteit amsterdam |
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Structure, function and evolution of nitric oxide reductase, an ancient oxidase (SENORA) | |
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Hierarchical control of denitrification | |
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The methylamine oxidation redox chain | |
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Period:1999-2001 |
Original research objectives
Many species of bacteria contain the enzyme nitric oxide reductase (NOR), which reduces nitric oxide (NO) to nitrous oxide. NOR belongs to the superfamily of terminal oxidases, which also includes the aa3-type terminal oxidase, responsible for the reduction of molecular oxygen to water and hence for much of biological energy metabolism in many organisms. By implication, their common ancestor of more than 2 billion years ago may have been a protein resembling NOR. Much of the structure and function of the terminal oxidases is known, but the catalytic mechanism of oxygen reduction and its coupling to proton pumping are not yet fully understood. This is partly because of a lack of functional enzymes with differences in the parameters that are critical for these properties. NOR may be such an enzyme: against the backdrop of the similarities in structure and function, NOR differs in two functional and two structural aspects from the oxidases. The functional differences are that NOR reduces NO at a higher rate than oxygen (whereas the oxidases reduce NO at a lower rate than oxygen) and that it appears to pump fewer if any protons. The structural differences are the presence of iron rather than copper in the binuclear metal center that is responsible for the reduction of oxygen or NO and the precise amino acid residues that reside in the alpha helices around the catalytic center. It was the objective of this proposal to determine whether these structural differences are responsible for the functional differences and hence generate a deeper insight into the structural evolution and catalytic mechanism of the superfamily of terminal oxidases. |
Partners
| Dr S Spiro Dr D Richardson School of Biological Sciences University of East Anglia Norwich NR4 7TJ UK PostDoc appointed: Technician appointed |
Professor M Saraste Postfach 10.2209 Meyerhofstrasse 1 69012 Heidelberg Germany PostDoc appointed: |
Dr S de Vries Delft University of Technology Faculty of Applied Sciences Kluyver Laboratory for Biotechnology Julianalaan 67 2628 BC Delft The Netherlands |
| Dr J Schouten MRC Holland Hudsonstraat 68 1057 SN Amsterdam The Netherlands |
Dr RJM van Spanning Free University Amsterdam De Boelelaan 1087 1081 HV Amsterdam The Netherlands PostDoc appointed: Technician appointed |
Professor C Varotsis University of Crete Department of Chemistry P.O. Box 1470 71409 Iraklion Crete Greece. |
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Original research objectives
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Neil Saunders, post doctoral fellow
Project: Hierarchical control of denitrification Period: 1998-2000 Present address: School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney 2052, NSW, Australia |
Marie Curie Research Fellowship. Project title "Hierarchical control of denitrification". Sub-projects: Structure and function of the NosR/NirI and NosX/NirX proteins of Paracoccus denitrificans, Transcriptional regulation of denitrification genes. It is the aim of the host institute to understand the hierarchical control of denitrification in soil bacteria (Paracoccus denitrificans is the model organism). In general, the four key enzymes of denitrification are expressed when the environmental oxygen concentration is low, and nitrate is available. Apparently, these two signals are the major triggers for the regulatory network as a whole. It has recently been found by the host institute that the oxygen concentration is monitored by a transcriptional activator designated FnrP. This protein belongs to a large family of FNR related proteins, some of which, like FnrP, contain an oxygen sensitive [4Fe-4S] cluster. During anaerobiosis, FnrP is active and promotes transcription of a number of genes amongst which is the nar gene cluster encoding nitrate reductase. Little is known about how the N-oxides are sensed in P. denitrificans. In E. coli, nar gene expression is, apart from FNR, under control of the NarXL and NarQP two component regulatory proteins, which sense the nitrate and nitrite concentrations.Knowledge on the control of expression of the nir, nor, and nos gene clusters encoding nitrite, nitric oxide, and nitrous oxide reductases, respectively, is emerging. Another member of the family of FNR-like transcriptional activators, designated NNR, specifically controls expression of the nir and nor gene clusters. Analyses of promoter-lacZ fusions have revealed that the promoters of the latter two gene clusters respond to nitrite and nitric oxide. Quite a few regulators other than NNR and FnrP, have been shown to exert control on the expression of these gene clusters as well. One of these proteins, designated NirI, is required for transcriptional activation of the nir gene cluster. The protein has transmembrane helices, a large periplasmic domain and a cytoplasmic domain harbouring cysteine motifs indicative for the presence of [4Fe-4S] clusters. The architecture of the protein makes it an ideal candidate for periplasmic signal sensing, and to transmit the signal via its cytoplasmic domain to as yet unknown transcriptional activation proteins. The gene encoding NirI is located upstream, and divergently transcribed from the nir gene cluster. A homologue of the nirI gene, designated nosR, has been encountered upstream of the nos gene cluster. At present, little is known about the function of its gene product, but the Pseudomonas stutzeri counterpart has been shown to be essential for transcription of the nos genes. |
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Period:1993-1995 |
Original research objectives
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'MADH REDOX CHAIN OF Thiobacillus versutus' EC NETWORK contract CHRX-CT93-0189 RESEARCH PLAN of CECILE DELORME
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Partners
| Prof G Sykes Dr C Dennison University of Newcastle UK |
Prof G canters Dr E Vijgeboom Leiden Institute of Chemistry Leiden University |
Prof H Duine Dr S de Vries Delft University of Technology Faculty of Applied Sciences Kluyver Laboratory for Biotechnology Julianalaan 67 2628 BC Delft The Netherlands |
| Dr LF Oltmann Dr RJM van Spanning Free University Amsterdam De Boelelaan 1087 1081 HV Amsterdam The Netherlands PostDoc appointed: Cecile Delorme |
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© November 4 2001 Rob van Spanning Home Page Rob van Spanning webmaster: Daoud Sie |
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