Purine nucleotides and their derivatives are ubiquitous molecules that serve many purposes in cells. For example, purines serve as the building blocks for DNA and RNA, act as signaling molecules, and provide a mechanism for energy storage and transfer. These molecules are made de novo in a highly conserved pathway that consists of ten stepwise reactions that convert phosphoribosyl pyrophosphate to inosine 5′-monophosphate. In general, prokaryotes tend to use freestanding single-functional enzymes for the chemical transformation, while the higher eukaryotes rely on multifunctional enzymes in this pathway. To date, investigations of the individual enzymes have revealed much about their respective mechanism of action. Until recently, however, there was little evidence to substantiate the attractive hypothesis that all of these enzymes act within a multi-enzyme complex framework.
Our discovery that all of the enzymes within the de novo purine biosynthetic pathway colocalize and assemble into a dynamic multi-enzyme complex has changed the way we think about the organization of metabolic pathways and validated a decades old hypothesis [1]. We have demonstrated that this protein assembly, called the purinosome, forms under conditions of purine starvation and can be modulated by extracellular nutrient levels and therapeutic intervention in cells [2-4, 6-7]. Initial characterization of the purinosome showed that de novo purine biosynthetic enzymes can be classified as either scaffold (PPAT, TGART, FGAMS) or periphery (PAICS, ASL, ATIC) based on their transient nature. Additional proteins such as adenylosuccinate synthetase, IMP dehydrogenase, and Hsp90 were also shown to interact with the purinosome and facilitate purine biosynthesis [6, 9]. We have also analyzed the morphological and temporal characteristics of the purinosome [8]. The greatest degree of purinosome formation was observed when cells are in the G1 phase of the cell cycle, which corresponds to when the cell is in highest purine demand. Furthermore, we have also observed enhanced flux of metabolites through the de novo purine biosynthetic pathway under cellular conditions that govern purinosome formation [9].
We have also shown that purinosomes spatially organize near mitochondria and interact with microtubules [3]. Current research projects are focused on exploring purinosome recruitment and dynamics at these subcellular foci. First, we are investigating the molecular factors that serve as biochemical triggers that initiate complex formation and facilitate recruitment to the mitochondria or microtubules. We employ a variety of biochemical, biophysical, and proteomic techniques to start to enhance our molecular understanding of how these enzyme clusters form. Second, we aim at understanding the impact that subcellular localization has on purinosome dynamics using stochastic optical reconstruction microscopy (STORM) in collaboration with Professor Xiaowei Zhuang at Harvard University. Our detailed characterization of the purinosome promises to answer pivotal questions regarding how enzyme clusters in metabolic pathways organize and provides a novel target that can be exploited for drug development for diseases such as cancer and rheumatoid arthritis.
Recent Publications
- An, S., R. Kumar, E. D. Sheets and S. J. Benkovic (2008). “Reversible compartmentalization of de novo purine biosynthetic complexes in living cells.” Science 320(5872): 103-106.
- An, S., Y. Deng, J. W. Tomsho, M. Kyoung and S. J. Benkovic (2010). “Microtubule-assisted mechanism for functional metabolic macromolecular complex formation.” Proc Natl Acad Sci U S A 107(29): 12872-12876.
- French, J. B., H. Zhao, S. An, S. Niessen, Y. Deng, B. F. Cravatt and S. J. Benkovic (2013). “Hsp70/Hsp90 chaperone machinery is involved in the assembly of the purinosome.” Proc Natl Acad Sci U S A 110(7): 2528-2533.
- Fu, R., D. Sutcliffe, H. Zhao, X. Huang, D. J. Schretlen, S. Benkovic and H. A. Jinnah (2015). “Clinical severity in Lesch-Nyhan disease: The role of residual enzyme and compensatory pathways.” Mol Genet Metab 114(1): 55-61.
- Chan, C. Y., H. Zhao, R. J. Pugh, A. M. Pedley, J. French, S. A. Jones, X. Zhuang, H. Jinnah, T. J. Huang and S. J. Benkovic (2015). “Purinosome formation as a function of the cell cycle.” Proc Natl Acad Sci U S A.
- Zhao, H., C. R. Chiaro, L. Zhang, P. B. Smith, C. Y. Chan, A. M. Pedley, R. J. Pugh, J. B. French, A. D. Patterson and S. J. Benkovic (2015). “Quantitative Analysis of Purine Nucleotides Indicates Purinosomes Increase de Novo Purine Biosynthesis.” J Biol Chem.
- French, J. B., Jones, S.A., Deng, H., Hu, H., Pugh, R. J., Chan C. Y., Kim, D., Pedley, A. M., Zhao, H., Zhang, Y., Huang, T. J., Fang, Y., Zhuang, X., and Benkovic, S. J., (2016) “Spatial colocalization and functional link of purinosomes with mitochondria ”, Science, 351:6274, 733-736.
Reviews
- Pedley, A. M. and Benkovic, S.J. (2016) “A new view into the regulation of purine metabolism – the purinosome”, Trends in Biochemical Sciences, doi.org/10/1016/j.tibs.2016.09.009.
- Zhao, H., J. B. French, Y. Fang and S. J. Benkovic (2013). “The purinosome, a multi-protein complex involved in the de novo biosynthesis of purines in humans.” Chem Commun (Camb) 49(40): 4444-4452.
Group Members
- Dr. Gargi Bhattacharyya
- Dr. Tony Pedley
- Dr. Vidhi Pareek
- Dr. Jingxuan He
- Dr. Yubing Liu
- Dr. Ling-Nan Zou
Collaborators
- Hyder (Buz) A. Jinnah, M.D., Ph.D., Emory University
- Dr. Xiaowei Zhuang, Harvard University
- Dr. Andrew Patterson, The Pennsylvania State University