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TECHNOLOGY ELUSIVE Pyrrolysine was first observed in the crystal structure of a methyltransferase enzyme, but the amino acid's exact structure has remained a mystery until now.
BUG'SOENETtC CODE ALLOWS PYRROLYSINE The 22nd amino acid is biosynthesized directly, making possible expansion of genetic code
L-pyrrolysine [Chem. Biol., 11,1317(2004)]. With chemically synthesized pyrroly sine in hand, both the Soil and Krzycki labs set about testing whether the amino acid's tRNA prefers to bind lysine (as would be ex pected if pyrrolysine biosynthesis parallels selenocysteine biosynthesis) or pyrrolysine (as would be expected if pyrrolysine is biosynthesized in the same way the stan dard 20 amino acids are). To their surprise, both groups independently found that pyrrolysine (but not lysine) can be attached directly to the pyrrolysine tRNA by an en zyme they dub pyrrolysyl-tRNA syn thetase. These results suggest that pyrroly sine is biosynthesized as a free amino acid and then attached to its tRNA. "This has never been seen before outside of the stan dard 20 amino acid set," Krzycki says.
THE UNEXPECTED result made Krzycki wonder whether his lab might be able to ex dle of the methyltransferase gene and that pand an organism's genetic code to accom the 22nd genetically encoded it directs the protein-making machinery to modate pyrrolysine simply by introducing amino acid, pyrrolysine, is insert pyrrolysine instead of stopping. A sim the genes for pyrrolysine tRNA and pyrrolybiosynthesized as a free amino ilarly reassigned codon is used for selenosyl-tRNA synthetase and chemically syn acid, something that hadn't been cysteine, the 21st amino acid. thesized pyrrolysine. As he had hoped, it seen before outside of the "standard" 20 Previous work had suggested that worked. Bacteria containing the genes for amino acids. This revelation has allowed an pyrrolysine was not likely to be synthesized the synthetase and the tRNA—as well as Ohio State University team to add pyrroly as a free amino acid, as the 20 standard the methyltransferase enzyme that normally sine—thus far found only in an anaerobic amino acids are. Instead, Krzycki's lab fig contains pyrrolysine—incorporate chemi microbe —to the genetically encoded ured pyrrolysine biosynthesis would paral cally synthesized pyrrolysine into amino acids that the model bacterium Es lel that of selenocysteine: A lysine the methyltransferase enzyme in cherichia coli uses to make proteins. would be attached to the pyrroly the correct position with high ef sine tRNA and then derivatized "Our study reveals that the natural world ficiency [Nature, 431,333 (2004)]. to give pyrrolysine. But recent re has used the same sort of strategy to ex Krzycki points out that nature's sults from the Ohio researchers, pand the genetic code that chemists have strategy to include pyrrolysine in as well as independent work from used in the laboratory to artificially expand the genetic code parallels the one Dieter Soil's lab at Yale Universi the genetic code for biotechnological ap chemists have used to expand the ty, have suggested otherwise. plications," says Ohio State microbiologist genetic code to produce proteins Joseph A. Krzycki, who worked with To figure out how the new containing novel amino acids: An chemist Michael K. Chan on the study. amino acid is incorporated into artificial synthetase enzyme is cre Chan's lab discovered pyrrolysine — an proteins, both the Ohio and Yale NHo ated that attaches a novel amino amide-linked 4-substituted pyrroline 5teams needed to make pyrroly Pyrrolysine acid to an artificial tRNA that in carboxylate lysine derivative—in the crys sine, a task made more difficult serts the novel amino acid at a re tal structure of a microbial methyltransby the fact that Chan's group ini assigned stop codon. ferase enzyme [Science, 296,1462 (2002)]. tially could not tell whether the 4-subThe 21 known genetically encoded amino stituent was a methyl, hydroxyl, or amino Such artificial synthetase-tRNA pairs acids are inserted into growing peptide group. Soil's team elected to synthesize have proven valuable for creating proteins chains by a dedicated transfer RNA (tRNA) racemic pyrrolysine with a methyl sub containing novel amino acids, but "no nat that recognizes a specific three-nucleotide stituent, having failed to make the hydroxyl ural examples were known to exist," note codon in the messenger RNA transcript. version [Proc. Natl. Acad. Sci. USA, 101, Paul Schimmel and Kirk Beebe of Scripps By isolating a pyrrolysine-specific tRNA, 12450 (2004)]. Chan's team, however, used Research Institute in an accompanying Na Krzycki's lab confirmed that pyrrolysine is an elegant set of chemical modification, ture commentary "In some ways, this dis genetically encoded, too [Science, 296,1459 crystallography, and mass spectrometry ex covery reaffirms that notion that, if some (2002)]. Earlier, his group had shown that periments to prove that pyrrolysine's 4thing can be done by experiment, it has a codon that's normally used to signal the substituent is a methyl group, then devel probably been done earlier by nature," they end of a gene is located smack in the mid oped a way to chemically synthesize note.—AMANDA YARNELL
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ECENT WORK HAS SHOWN THAT
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