Progress 12/01/09 to 11/30/12
Outputs
OUTPUTS: The blue copper oxidase (BCO) and nitrite reductase enzymes from Nitrosomonas europaea were characterized by X-ray crystallography and biochemical assays. New methods for detecting multicopper oxidase activity in gels were developed. We also participated in annotation of the genome of the archaeal nitrifier N. maritimus. Finally, a soluble protein corresponding to part of the N. yellowstonii ammonia monooxygenase (AMO) was developed as a model system. Our findings were presented at the Gordon Research Conference on the Molecular Basis of Microbial One-Carbon Metaoblism in August 2012, the Graduate Research Seminar in Bioinorganic Chemistry in January 2012, the 2nd International Conference on Nitrification (ICoN2) in July 2011, the Gordon Research Conference on the Molecular Basis of Microbial One-Carbon Metabolism in August 2010, and the 1st International Conference on Nitrification in 2009. The body of work generated by this project constitutes the doctoral thesis of Thomas Lawton. PARTICIPANTS: The PI, Amy C. Rosenzweig, guided all aspects of the project, including designing experiments, writing and editing manuscripts, and preparing oral and poster presentations. Parts of the research were conducted in collaboration with Daniel Arp and his laboratory. A graduate student, Thomas Lawton, conducted most of the experiments and is preparing his doctoral dissertation on this project. In addition, Thomas worked with several Northwestern undergraduate students, including Kimberly Bowen, Jungwha Ham, and Tianlin Sun. This project thus provided both doctoral training and undergraduate research experiences. Moreover, the graduate student, Thomas Lawton, gained valuable experience in advising and directing undergraduate researchers. All of these undergraduate students also were awarded internal Northwestern University summer funding to work on this project during the summers of 2010, 2011, and 2012. TARGET AUDIENCES: This project has provided a valuable training experience for Northwestern undergraduate students. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The crystallographic data generated by this project contribute to understanding the molecular basis for microbial nitrogen metabolism. In addition, these data contribute to the fundamental understanding copper protein evolution New protocols for isolating and determining the activities of proteins from nitrifying bacteria have also resulted and will impact future work involving biochemistry in native organisms. A graduate student and several undergraduate students received valuable research training.
Publications
- Lawton, T. J.; Rosenzweig, A. C. Two-domain multicopper oxidase. In Handbook of Metalloproteins Volumes 4 and 5 (A. Messerschmidt, ed.), 2011, John Wiley & Sons, Chichester, UK, 591-599, originally published online September 2010.
- Walker, C. B.; de la Torre, J. R.; Urakawa, H.; Klotz, M. G.; Lawton, T. J.; Pinel, N.; Arp, D. J.; Brochier-Armanet, C.; Chain, P. S. G.; Chan, P. P.; Golabgir, A.; Hemp, J.; Hugler, M.; Karr, E. A.; Konneke, M.; Shin, M.; Lawton, T. J.; Martens-Habbena, W.; Sayavedra-Soto, L. A.; Lang, D.; Sievert, S. M.; Rosenzweig, A. C.; Manning, G.; Stahl, D. A. The Nitrosopumilus maritimus genome reveals unique mechanisms for nitrification and autotrophy in globally distributed marine crenarchaea. Proc. Natl. Acad. Sci. USA 2010, 107, 8818-8823.
- Lawton, T. J.; Rosenzweig, A. C. Crystal structure of apo-amoB from Nitrosocaldus yellowstonii, 2013, in preparation.
- Lawton, T. J.; Sayavedra-Soto, L.; Arp, D. J.; Rosenzweig, A. C. Crystal structure of a unique nitrite reductase, 2013, in preparation.
- Lawton, T. J.; Rosenzweig, A. C. Detection and characterization of a multicopper oxidase from Nitrosomonas europaea. Methods Enzymol. 2011, 496, 423-433.
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Progress 12/01/10 to 11/30/11
Outputs
OUTPUTS: We have completed our structural characterization of nitrite reductase from N. europaea. We have solved multiple structures, including at different pH values and different redox states of the copper ions. We are currently preparing these data for publication. This work was presented at the 2nd International Conference on Nitrification (ICoN2) in July 2011 in Nijmegen, The Netherlands. We have continued to work on the ammonia to nitrite oxidizing system. PARTICIPANTS: A graduate student, Thomas J. Lawton, has conducted all the experimental work for this project. He has obtained valuable training in writing up data for publication and preparing poster and oral presentations. We continue to collaborate closely with Professor Dan Arp at Oregon State University. The principal investigator, Amy Rosenzweig, has overseen all aspects of the project. TARGET AUDIENCES: This project has provided a learning experience for multiple undergraduate students. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The structural characterization of nitrite reductase contributes to understanding the physiological role of this enzyme in nitrifying bacteria as well as to a general picture of copper protein evolution. Several protein purification and assay techniques were developed while isolating the various target proteins from native N. europaea bacteria.
Publications
- Lawton, T. J.; Rosenzweig, A. C. Detection and characterization of a multicopper oxidase from Nitrosomonas europaea. Methods Enzymol. 2011, 496, 423-433.
- Lawton, T. J.; Rosenzweig, A. C. Two-domain multicopper oxidase. In Handbook of Metalloproteins Volumes 4 and 5 (A. Messerschmidt, ed.), 2011, John Wiley & Sons, Chichester, UK, 591-599, originally published online September 2010.
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Progress 12/01/09 to 11/30/10
Outputs
OUTPUTS: We have made progress toward characterizing the ammonia to nitrite oxidizing system. We have optimized the isolation of ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO). We have developed methods to alleviate protein aggregation problems and have improved the stability of our isolated samples. For the nitrite reduction system, we have determined the crystal structure of nitrite reductase and are conducting biochemical assays. Purification of other gene products encoded by the nitrite reductase gene cluster is underway. Several new methods to interrogate protein-protein interactions have been established in the laboratory. PARTICIPANTS: A graduate student, Thomas J. Lawton, is working on all aspects of this project. The PI, Amy C. Rosenzweig, is overseeing all aspects of this project. We continue to collaborate with Daniel Arp (Oregon State University). Thomas J. Lawton has been aided by several undergraduate researchers. TARGET AUDIENCES: This project has provided an opportunity for undergraduate students at Northwestern to gain research experience and learn practical laboratory skills. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The nitrite reductase molecular structure is now known in detail. Several new laboratory methods and techniques have been developed, including a gel based activity assay for multicopper oxidases (Methods Enzymol., in press).
Publications
- Lawton, T. J.; Rosenzweig, A. C. Detection and characterization of a multicopper oxidase from Nitrosomonas europaea. Methods Enzymol. 2011, in press.
- Lawton, T. J.; Rosenzweig, A. C. Two-domain multicopper oxidase. Handbook of Metalloproteins (A. Messerschmidt, ed.) 2011, in press.
- Walker, C. B.; de la Torre, J. R.; Urakawa, H.; Klotz, M. G.; Lawton, T. J.; Pinel, N.; Arp, D. J.; Brochier-Armanet, C.; Chain, P. S. G.; Chan, P. P.; Golabgir, A.; Hemp, J.; Hugler, M.; Karr, E. A.; Konneke, M.; Shin, M.; Lawton, T. J.; Martens-Habbena, W.; Sayavedra-Soto, L. A.; Lang, D.; Sievert, S. M.; Rosenzweig, A. C.; Manning, G.; Stahl, D. A. The Nitrosopumilus maritimus genome reveals unique mechanisms for nitrification and autotrophy in globally distributed marine crenarchaea. Proc. Natl. Acad. Sci. USA 2010, 107, 8818-8823.
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