news of the week nature of a critical catalytic intermediate,” says Paul R. Ortiz de AlkB Montellano of the University of Cys129 Fe(II) California, San Francisco. “Most of αKG the earlier work involved indirect S ENZYMOLOGY: Independent teams measurements and were useful but characterize key intermediates S imprecise. Given the importance in cellular oxidations of cytochrome P450 in biology, medicine, and biotechnology, an understanding of the basic physical ORKING INDEPENDENTLY and after years and chemical properties of its cataof unsuccessful efforts, two groups have caplytic species is a major advance.” CLOSE ENCOUNTER He and tured and characterized critical intermediates Intermediates of DNA-repair coworkers linked DNA substrates in oxidation reactions catalyzed by two key enzyme dioxygenases also could not be bearing modified bases (red oval) to families: cytochrome P450 monooxygenases and DNAobserved before, because they cysteine-129 in AlkB, immobilizing repair dioxygenases. are unstable in water after release each base in the active site. They The P450 monooxygenase study helps confirm from an enzyme. To capture AlkB then crystallized the complexes researchers’ understanding of the workings of a ubiqui- catalytic intermediates, Chuan with Fe(II) and α-ketoglutarate tous group of enzymes that play a critical role in nutriHe of the University of Chicago; (αKG) cofactors and added oxygen ent and drug metabolism. And the DNA-repair dioxyQiang Cui of the University of to initiate oxidative demethylation genase report helps nail down the detailed molecular Wisconsin, Madison; and coworkof each substrate. The reaction mechanism of oxidative demethylation, a process that ers cross-linked each of three froze at the intermediate stage, plays a major role in epigenetic gene programming and modified-DNA substrates of AlkB enabling the team to observe and in conditions such as obesity and cancer. to its active site. They crystallized characterize the intermediates. Cytochrome P450 enzymes incorporate a heme iron the complexes with cofactors iron center and use O2 to hydroxylate aliphatic carbon-hydro- and α-ketoglutarate but without gen bonds, with the remaining oxygen reduced to water. oxygen. Adding oxygen to the crystals caused oxidative Researchers have tried for decades to pin down a key indemethylation of each substrate to begin but to stop termediate in P450 catalysis, a postulated iron(IV)-oxoearly, enabling crystallographic characterization of the porphyrin cation radical known as compound I. intermediates (Nature, DOI: 10.1038/nature09497). Jonathan Rittle and Michael T. Green of PennsylGregory Verdine of Harvard University first used vania State University finally trapped compound I by disulfide cross-linking to capture conformational interusing a P450 enzyme from a species of thermophilic mediates in DNA repair but notes that it has not been archaea, a peroxide substrate, and rapid freeze-quench used before “to poise a complex in a precatalytic state techniques (Science, DOI: 10.1126/science.1193478). and then observe reaction intermediates.” The intermeTheir electronic and kinetic characterization of the diates He and coworkers found had been hypothesized species confirms that it is, in fact, an iron(IV)-oxo com- earlier, “but other mechanisms and structures were pospound and can perform the hydroxylation reaction. sible,” Verdine says, adding that the study “provides a “This is an excellent piece of work that clarifies the detailed glimpse into the action of one dioxygenase and by extension the class at large.” This is “the next big step in understanding DNA and RNA oxidative demethylation,” says John D. Lipscomb of the University of Minnesota, who has used enzyme crystals to H O O study oxygen binding. “It removes a great deal H of speculation about the intermediates and FeIII FeIV establishes a likely prototype for all of the en• S S zymes from this large and growing family that modify or remove groups from DNA, RNA, and histones. Finally, this is an excellent demonstration of the remarkable ability of enzyme LONG-SOUGHT Cytochrome P450 enzymes react with O2 crystals to slow or stop reactions mid-cycle so to form water and compound I (left), an iron(IV)-oxo-porphyrin actual intermediates can be obtained.”—STU radical species, which then hydroxylates C–H bonds.
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