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Functional context, biosynthesis, and genetic encoding of pyrrolysine

Marsha A Gaston1, Ruisheng Jiang, Joseph A Krzycki

  • 1Department of Microbiology, 484 West 12th Avenue, The Ohio State University, Columbus, OH 43210, United States.

Current Opinion in Microbiology
|May 10, 2011
PubMed

Insights

Pyrrolysine, the 22nd amino acid, is biosynthesized by specific genes (pyl) in some microbes. These genes enable pyrrolysine incorporation into proteins, offering new possibilities for protein engineering.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Microbiology

Background:

  • Amber codons in methylamine methyltransferase genes are translated as pyrrolysine (the 22nd amino acid) in Methanosarcina spp.
  • Pyrrolysine biosynthesis (pyl) genes and pyrrolysine-containing methyltransferase genes are found in diverse archaeal and bacterial genera, including human pathogens.

Purpose of the Study:

  • To elucidate the pyrrolysine biosynthetic pathway in Escherichia coli.
  • To characterize the pyrrolysyl-tRNA synthetase (PylS) and its role in pyrrolysine incorporation.
  • To explore the potential of the pyrrolysine system in recombinant protein engineering.

Main Methods:

  • Genetic identification of pyl genes in various genera.
  • Recombinant expression of pylBCD and pylTS genes in Escherichia coli.
  • Biochemical characterization of pyrrolysine biosynthesis pathway intermediates.
  • Structural analysis of pyrrolysyl-tRNA synthetase (PylS).

Main Results:

  • Pyrrolysine is synthesized from lysine via methylornithine by PylB, PylC, and PylD.
  • The PylS enzyme (or bacterial PylSc) binds both pyrrolysine and tRNAPyl.
  • Pyrrolysine incorporation into proteins is directed by the pylTS gene products.

Conclusions:

  • The complete pyrrolysine biosynthetic pathway and its genetic basis have been identified.
  • The pyrrolysyl-tRNA synthetase structure provides insights into pyrrolysine activation and transfer.
  • The pyrrolysine system (PylS and tRNAPyl) represents a valuable orthogonal pair for introducing non-canonical amino acids into proteins.

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