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Area of general research interest:
The Structure and biochemistry of nitric oxide synthases
Current program:
- Determining how enzyme structure influences catalysis.
- Investigating the mechanisms by which substrate and calmodulin control electron transfer between enzyme redox centers.
- Determining the role of tetrahydrobiopterin and heme in oxygen activation by NDS.
Investigators:
- Dennis J. Stuehr, Ph.D.
- Subrata Adak, Ph.D.
- Kulwant Singh Aulak, Ph.D.
- John McDonald
- David Konas, Ph.D.
- Thomas Koeck, Ph.D.
- Jerome Santolini, Ph.D.
- Koustubh Panda, Ph.D.
- Manisha Sharma
- Zhiqiang Wang, Ph.D.
- Chin-chuan Wei, Ph.D.
Collaborators:
- Getzoff, Elizabeth, Ph.D., Scripps Research Institute, La Jolla, CA
- Rousseau, Denis, Ph.D., AT & T Bell Labs, Summitt, NJ
- Brudvig, Gary, Ph.D., Yale University, CT
Brief Description:
Our laboratory studies nitric oxide (NO) biosynthesis in mammals. Discovered in 1987, this novel biochemical pathway is now considered to be a widespread means for regulation of cell function and communication. Specific systems in which the pathway participates are signal transduction in the brain, stroke, control of blood pressure and heart rate, gastric motility, and immunologic destruction of tumor cells and microbes. Our main interests are in uncovering the enzyme reaction mechanism, understanding how enzyme structure relates to function, learning how other cellular proteins can control NOS activity, and the cell biology of NO as it relates to gene expression and protein nitration.
Reaction mechanism and structure-function.
Biosynthesis of NO is carried out by enzymes named nitric oxide synthases (NOSs). Our lab works with three distinct NOS: A cytokine-induced macrophage isoform and two constitutively expressed NOS from neurons and endothelium. All NOS isoforms catalyze a reaction that is complex and biochemically novel: the conversion of L-arginine to nitric oxide and citrulline. We are currently working out the reaction mechanism of NOS through a variety of strategies. These include utilizing chemically modified substrates, intermediates, and cofactors, techniques such as Raman and electron paramagnetic spectroscopy, crystallography, rapid mixing stopped flow technology, and single turnover studies to probe the enzyme's active site chemistry. We also are analyzing important domains within the enzyme (for example, those involved in calmodulin, flavin, or heme binding) by site-directed mutagenesis and crystallography. By crystallography we already have visualized the active center of the NOS and identified regions in the protein that enable two NOS subunits to interact and bind its cofactors and substrate. We are studying why a dimeric interaction is required to activate the enzyme, and have found it allows electron transfer between two NOS subunits in the active dimer. Ongoing mutagenesis projects are based on our crystallographic information and include mapping the surface residues that allow for electron transfer between the two subunits of NOS, identifying catalytically important residues in the active site, and residues that are important in transducing the effects of bound essential cofactors such as tetrahydrobiopterin.
Assembly, activation, control of NO synthases.
The NOS are controlled at multiple levels. We are studying how the active form of the enzyme is assembled within cells. At this point we know that intracellular heme availability determines how much of NOS is assembled into its active form, and that NO produced by the enzyme can somehow prevent heme incorporation into the protein. Our understanding of assembly regulation may help develop a new means to control NO synthesis during inflammation. Another area of research involves activation of the neuronal and endothelian NOS by calcium. Two steps in the NOS electron transfer sequence are controlled by the calcium binding protein named calmodulin. Calmodulin enables electrons to pass to the iron atom, and also greatly speeds the introduction of electrons into the enzyme's flavin groups. We are now studying how calmodulin attachment changes the enzyme's structure to allow these changes in electron transfer, using rapid spectroscopic techniques, and mutants of calmodulin and NOS.
Cell biology.
We are studying three aspects of NOS regarding cell biology. One project seeks to understand how NOS interactions with other cellular proteins such as caveolins or kalirin modulate the assembly and activity of the NOS enzymes at a molecular level. Another project is focused on nitration of cellular proteins during NO synthesis--what components participate, how nitration can be selectively blocked, whether nitration causes change in protein function or gene expression, and whether enzymes exist that can remove or reduce the nitro group from proteins. A third project is investigating gene expression changes in response to NO synthesis under different cellular conditions, keying on enzymes involved in iron uptake, storage, and homeostasis.
Key References:
Wei, C.-C., Z. wang, Q. Wang, A. L. Meade, C. Hemann, R. Hille, and D. J. Stuehr. 2000. Rapid kinetic studies link tetrahydrobiopterin radical formation, heme-dioxy reduction, and arginine hydroxylation in inducible NO synthase. J. Biol. Chem., 276, 315-319
Adak, S., Santolini, J., Tikunova, S., Wang, Q., Johnson, J. D., and Stuehr, D. J. 2001. Neuronal NO synthase mutant (S1412D) demonstrates surprising connections between heme reduction, NO complex formation, and catalysis. J. Biol. Chem., 276, 1244-1252
Adak, S. and D. J. Stuehr. 2001. A proximal tryptophan in NO synthase controls activity by a novel mechanism. J. Inorg. Biochem. 83, 301-308
Panda, K., Ghosh, S., and Stuehr, D. J. 2001. Calmodulin activates intersubunit electron transfer in the neuronal NO synthase dimer. J. Biol. Chem., 276, 23349-56.
Aoyagi M, Arvai AS, Ghosh S, Stuehr DJ, Tainer JA, Getzoff ED. (2001) Structures of tetrahydrobiopterin binding-site mutants of inducible nitric oxide synthase oxygenase dimer and implicated roles of trp457(,). Biochemistry 40,12826-32.
Wang ZQ, Wei CC, Ghosh S, Meade AL, Hemann C, Hille R, Stuehr DJ. (2001) A conserved tryptophan in nitric oxide synthase regulates heme-dioxy reduction by tetrahydrobiopterin. Biochemistry 40, 12819-25.
Aulak KS, Miyagi M, Yan L, West KA, Massillon D, Crabb JW, Stuehr DJ. (2001) Proteomic method identifies proteins nitrated in vivo during inflammatory challenge. Proc. Natl. Acad. Sci. USA 98, 12056-61.
Santolini J, Meade AL, Stuehr DJ. (2001) Differences in three kinetic parameters underpin the unique catalytic profiles of NOS I, II, and III. J. Biol Chem. 276, 48887-98
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