Functional context, biosynthesis, and genetic encoding of pyrrolysine

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.