By David N. Leff

Last Thursday, April 19, 2001, NASA flight controllers pointed the Mars Odyssey spacecraft¿s imaging system at the Earth and moon, to calibrate its cameras. They were looking, among other things, for sediment that could have been deposited by water.

Sure signs of water on the Red Planet would likely cinch the suspicion that Mars has ¿ or once had ¿ some form of life on its surface. Another salient witness to living organisms on that planet would be traces of amino acids.

On our own planet, these familiar organic molecules are known for their ubiquitous role in managing the genetic code¿s task of threading DNA¿s triplet codons through a cell¿s ribosomes, via transfer RNA, and on to connecting downstream amino acids into peptide, polypeptide and protein chains. Amino acids get attached to a transfer RNA in the first step of protein synthesis.

In this process, life on earth employs precisely 20 amino acids. These ¿essential¿ amino acids equip every single life form, from the archaea heat-and-cold-loving bacteria on up the ladder of life via prokaryotic parasites to eukaryotic worms, insects, birds and mammals ¿ including us.

These essential amino acids share one identity badge ¿ they are optically levo-rotary, ¿L¿ for short. On the other hand, most of the ¿unnatural¿ amino acids, with no place at life¿s table, go in for right-handed amino acids ¿ dextro-rotary, or ¿D.¿

There are literally hundreds of these non-standard molecules, synthesized by nature or in cells of living organisms. If and when amino acids ¿ offbeat or essential ¿ turn up on Mars, they will share an earthly ancestry that goes back millions of years to the pre-Cambrian geologic era. At least 22 fossil amino acids have been discovered so far in its ancient rocks.

Two Ways To Defeat The ¿Essential¿ Number

To many organic biochemists, the 20-essential-amino-acid limit seems like a molecular strait-jacket. Many are trying to pry open this constraint, and insert non-conventional add-ons, in hopes of generating new and useful proteins.

Two such research groups are featured in the current issue of Science, dated April 20, 2001. Both are on the faculty of the Scripps Research Institute in La Jolla, Calif., but each is independent of the other. One article bears the title: ¿Enlarging the amino acid set of Escherichia coli by infiltration of the valine coding pathway.¿ Its joint corresponding authors are molecular biologist and biochemist Paul Schimmel of Scripps, and Philippe Marliere, at Genoscope, France¿s National Genome Sequencing Centre, in the Parisian suburb of Evry.

The second paper is titled: ¿Expanding the genetic code of Escherichia coli.¿ Its senior author is chemical biologist Peter Schultz, and its lead author, bioorganic chemist Lei Wang, a graduate student in the Schultz lab.

¿I think we have developed a general methodology,¿ Wang told BioWorld Today, ¿to put unnatural amino acids site-specifically into proteins in a living cell ¿ in our case right now, E. coli. This is the first time,¿ he added, ¿that people have put unnatural amino acids into proteins site-specifically in vivo.¿

Contrasting the two papers, Wang made the point, that, ¿The uniqueness of our paper is that we are adding a new, synthetic amino acid ¿ with tRNA and synthetase developed from an archaea bacterium ¿ into the proteins of E. coli. It is O-methyl L tyrosine, which is very similar to the natural amino acid, tyrosine, but is not found in any living organism. And in our first experiment, we succeeded in getting the bug to take it.¿ The co-authors forced their construct into the bacterium¿s cell ¿by a pretty conventional method ¿ high-voltage electrophoresis.¿

Schimmel and his co-authors took a different tack. ¿What¿s interesting in our work,¿ he said, ¿is that we put a transfer-RNA/synthetase point mutation into the editing site of synthetases. This is one of the proteins that decode genetic information in the cell. We found that that single change ¿ from a threonine 222 to a proline ¿ resulted in all the gene products in the cell being changed.¿

Killing The Proofreader Did It

¿We knew that this region of the protein encodes the editing site, a site that clears out mistakes in amino acylation before it gets into a growing polypeptide. What we did was disable that editing site so it couldn¿t get rid of the mistakes. Then we introduced alpha-amino-butyrate ¿ an amino acid that¿s not found in proteins. We replaced about a quarter of all the valines in all the proteins in E. coli. We had invaded the genetic code, showing that one can produce novel polypeptide-based biomaterials with all sorts of new amino acids.¿

On Schimmel¿s drawing board is a far-out project ¿ ¿to try to even eliminate one of the 20 amino acids from E. coli, and see whether we can get cells that have only 19 essential amino acids. This would be of great theoretical interest to people who are studying evolution and the origin of life. But I think it may also be possible to use such an organism as a starting point to introduce another kind of amino acid, and bring it back to 20. And that 20th amino acid would be totally different than one ever used before.

¿These non-20s,¿ Schimmel explained, ¿are by-products of normal cell metabolism. Some of them are not found in the cell normally, others are. Some of them occur in nature, in other organisms, and can be made chemically by synthetic methods.

¿Because we have shown how drastic the effect of a single editing-site point mutation is,¿ Schimmel observed, ¿we would certainly imagine that it may be worthwhile to investigate the possibility that such mutations occur naturally in humans ¿ and may be associated with specific metabolic diseases. For people who are interested in new polypeptide-based biomaterials, this could be of potential long-term interest.¿

Schultz and Lei Wang also foresee industrial uses, for example, stabilizing enzymes, degrading industrial wastes, and, Wang suggested, ¿finding better drugs, because you can get new properties of proteins by introducing unnatural amino acids. We are also working on mammalian cells; E. coli is just a bacterial prokaryote. We can also alter eukaryotes, including yeasts and mammalian cells.¿

An accompanying ¿Perspective¿ in Science pointed out that, ¿The ability to engineer organisms with an expanded amino acid repertoire will be hugely beneficial to biotechnology.¿

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