COMPLEX SYNTHESIZES PURINES BIOCHEMISTRY: First evidence for
the existence of ‘purinosome’
S
CIENTISTS 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.” PURINE PATHWAY To find the complex, Benkovic The proposed purinosome protein and his coworkers—postdocs complex converts phosphoribosyl Songon An and Ravindra Kumar pyrophosphate to inosine and assistant professor Erin D. monophosphate. Sheets—fluorescently labeled pairs O O of the enzymes and expressed them O O in human cancer cells. They then O P O P OH HO P O used fluorescence microscopy to O– O– O– determine whether the enzymes HO OH form complexes. Initially, the enzymes were disSix enzymes persed throughout the cytoplasm. 10 reactions “We realized that we had to force O the cells to have a greater demand N NH for purines so that they would not O be using purines they could salvage N O N HO P O from the growth medium,” Benkovic says. When the researchers O– HO OH 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?” Enzymes in the purine biosynthetic “As someone who has worked on this pathway and pathway labeled believed in the importance of transient protein-protein with green and interactions,” Stubbe says, “I think the experiments are orange fluorescent very provocative and exciting.” proteins form Benkovic’s team so far has shown only that pairs of clusters when the enzymes colocalize. Now, using affinity chromatogcells are grown in purine-depleted raphy, they are trying to isolate the intact six-enzyme growth medium. complex. They also plan to do fluorescence resonance energy transfer experiments to obtain spatial informa-
PHARMACEUTICAL CHEMICALS Dowpharma sells its U.K. operations to India’s Dr. Reddy’s Dowpharma says it will sell its small-molecule active pharmaceutical ingredient (API) assets in Cambridge and Mirfield, England, to Dr. Reddy’s Laboratories, an Indian generic drug company that also runs a custom manufacturing business. Dow says the divestiture is part of a company-wide portfolio review and will not affect its API assets in Midland, Mich., or its Pfenex Pseudomonas-based protein expression technology business. Dr. Reddy’s will obtain a nonexclusive license to Pfenex as part of the agreement. “The businesses are doing well, but they are not a strategic fit for the
way Dow is run now,” says a company spokesman, noting that their process technology differs from that used in Midland and that raw materials are more expensive in Europe. Dow acquired the U.K. facilities with its purchase of Ascot’s Chirotech Technology and Mitchell Cotts fine chemicals operations in 2001. James Bruno, an independent fine chemicals and pharmaceutical consultant, sees the deal as evidence that Dow is following other diversified Western chemical companies out of the custom pharmaceutical chemical manufacturing business. “These were supposed
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to be their strategic sites,” Bruno says. The Dow spokesman denies this. “The Midland plant has a lot of long-term contracts that are operating very well,” he says. “We can build in more contracts if we want.” Dr. Reddy’s, which purchased an API plant in Mexico from Roche in 2005, is on a campaign to build a global custom manufacturing operation. “The proprietary chiral and biocatalysis technology at the Cambridge site and the scale-up capability in the Mirfield site will add significant value to the company,” Managing Director Satish Reddy says. —RICK MULLIN
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