May 27, 2002
Volume 80, Number 21
CENEAR 80 21 p. 13
ISSN 0009-2347



Methanogen uses stop codon
to genetically encode l-pyrrolysine


Nature uses only a handful of amino acid building blocks to generate immense complexity from a relatively simple genetic code. There are just 20 genetically encoded amino acids in mammalian cells; a few organisms use 21. Now, researchers at Ohio State University have discovered a 22nd [Science, 296, 1459 and 1462 (2002)].

The team, led by microbiology professor Joseph A. Krzycki and chemistry and biochemistry professor Michael K. Chan, calls the amino acid l-pyrrolysine. The novel residue is an amide-linked 4-substituted pyrroline-5-carboxylate lysine derivative.

Chan and graduate student Bing Hao identified pyrrolysine in the 1.55-Å crystal structure of monomethylamine methyltransferase (MtmB) from the microbe Methanosarcina barkeri, found at the bottom of freshwater lakes. An enzyme in the pathway that makes methane from methylamine, MtmB has a barrel protein fold with pyrrolysine tucked into the bottom.

NOVEL Pyrrolysine (right) was identified in a methyltransferase of a methane-generating microbe. The substituent at C-4 could not be conclusively determined from the electron density map.
But finding a novel amino acid in a protein structure doesn't necessarily mean the residue is genetically encoded. In the protein collagen, for example, 4-hydroxyproline and 5-hydroxylysine are introduced by modification of the appropriate amino acid after the polypeptide chain has been synthesized. To qualify as genetically encoded, amino acids must be directly inserted into the growing peptide chain by a dedicated transfer RNA (tRNA) that recognizes a specific three-nucleotide codon in the messenger RNA (mRNA) transcript.

Pyrrolysine seems to qualify. As in the incorporation of selenocysteine, the 21st amino acid, a codon normally used to signal the end of a gene "is taken over by a specialized tRNA which carries an aminoacyl residue developed from one of the canonical amino acids," explains microbiologist August Böck of the University of Munich.

Smack in the middle of the MtmB gene--right where the pyrrolysine-specific codon is expected to be--is the stop codon UAG. But this particular UAG doesn't stop MtmB translation. Instead, a full-length protein is produced.

Krzycki and graduate students Carey M. James and Gayathri Srinivasan found two other genes next to the MtmB gene. One gene encodes a specialized tRNA (tRNACUA) that recognizes the UAG stop codon in the mRNA transcript; the other, a novel aminoacyl tRNA synthetase that is capable of loading tRNACUA with lysine.

Lysine-loaded tRNACUA is likely an intermediate in pyrrolysine-tRNACUA formation, just as selenocysteine is assembled on its specialized tRNA by enzymatic modification of serine, one of the 20 "standard" amino acids.

The presence of this machinery "indicates that pyrrolysine is inserted into MtmB during translation," Krzycki says.

But the mechanism of pyrrolysine biosynthesis and incorporation is still uncertain, he cautions. His lab is currently trying to fish out tRNACUA carrying pyrrolysine from M. barkeri. Chan's lab is working to confirm the structure of pyrrolysine and to chemically synthesize pyrrolysine for labeling studies. It also remains to be seen how this subverted UAG codon is differentiated from UAG stop codons, which are found at the end of 5% of known M. barkeri genes.

Despite the unanswered questions, the papers "present an additional example of the flexibility of the genetic code," Böck says. "That there is a general way to incorporate nonstandard amino acids is very interesting from an evolutionary point of view."

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