Complex Synthesizes Purines | April 7, 2008 Issue - Vol. 86 Issue 14 | Chemical & Engineering News
Volume 86 Issue 14 | p. 13 | News of The Week
Issue Date: April 7, 2008

Complex Synthesizes Purines

First evidence for the existence of 'purinosome'
Department: Science & Technology
News Channels: Biological SCENE
Purine pathway
The proposed purinosome protein complex converts phosphoribosylpyrophosphate to inosinemonophosphate.
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Purine pathway
The proposed purinosome protein complex converts phosphoribosylpyrophosphate to inosinemonophosphate.
Enzymes in the purine biosynthetic pathway labeled with green and orange fluorescent proteins form clusters when cells are grown in purine-depleted growth medium.
Credit: Science © 2008
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Enzymes in the purine biosynthetic pathway labeled with green and orange fluorescent proteins form clusters when cells are grown in purine-depleted growth medium.
Credit: Science © 2008

SCIENTISTS AT Pennsylvania State University propose that a new protein complex they call the "purinosome" is responsible for the biosynthetic pathway that makes purines from scratch (Science 2008, 320, 103).

"The existence of the purinosome will stimulate experiments for understanding the molecular basis of its formation, the mechanism by which it accelerates purine biosynthesis, and the search for additional cellular components that regulate its formation," says Steven E. Ealick, a Cornell University chemistry professor studying purine biosynthesis who was not involved with the research. "The paper describes beautifully conceived experiments leading to results that will change the way in which researchers think about cellular metabolism."

In eukaryotic cells, the de novo purine biosynthetic pathway involves six enzymes that catalyze 10 reactions to form inosine monophosphate, the starting point for adenosine and guanosine nucleotides, from phosphoribosyl pyrophosphate. "There was a fair amount of reasoning that suggested that the six enzymes should be in some kind of complex," says Stephen J. Benkovic, the Penn State chemistry professor who led the research. Experiments in solution failed to provide compelling evidence of such a complex.

"Redundancy of function often makes it challenging to see organization," says JoAnne Stubbe, a biochemist at MIT who has studied the purine pathway in bacteria. "The purine pathway has many unstable intermediates, and some of these intermediates partition into different pathways."

To find the complex, Benkovic and his coworkers—postdocs Songon An and Ravindra Kumar and assistant professor Erin D. Sheets—fluorescently labeled pairs of the enzymes and expressed them in human cancer cells. They then used fluorescence microscopy to determine whether the enzymes form complexes.

Initially, the enzymes were dispersed throughout the cytoplasm. "We realized that we had to force the cells to have a greater demand for purines so that they would not be using purines they could salvage from the growth medium," Benkovic says. When the researchers grew the cells in purine-depleted medium, the enzymes formed clusters.

The formation of those clusters has turned out to be reversible. "The fact that we could make the cluster come and go opens a whole new area of inquiry," Benkovic says. "What makes it come and go? Is it the level of purines in the cell? Is there some kind of signaling that modifies the proteins by phosphorylation?"

"As someone who has worked on this pathway and believed in the importance of transient protein-protein interactions," Stubbe says, "I think the experiments are very provocative and exciting."

Benkovic's team so far has shown only that pairs of the enzymes colocalize. Now, using affinity chromatography, they are trying to isolate the intact six-enzyme complex. They also plan to do fluorescence resonance energy transfer experiments to obtain spatial information about the complex.

 
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