03‐327/727 2015 [Type text] Pieter Spealman

Introduction to Pyrrolysine

This year’s hands‐on practical assignments will focus on enzymes in the biosynthesis pathway for pyrrolysine, the 22nd amino acid. This introduction seeks to familiarize you with the general concepts of the genetic code and the enzymes a cell must possess in order to incorporate pyrrolysine into its genetic code.

The process of turning genes into proteins is, in part, the process of translating the sequence of nucleotides encoded in the messenger‐RNA (mRNA) into the sequence of amino‐acids that make up the protein. The key to this process is the role which transfer‐RNA (tRNA) plays. Triplets of the mRNA nucleotides, called codons, are matched (via Watson‐Crick base‐pairing) to the anti‐codons of a particular species of tRNA. Many species of tRNA exist – each species has a unique anti‐codon sequence and carries an amino‐acid specific to that anti‐codon. In addition to the codons that match tRNA, there are also codons without a matching tRNA. These are called Stop codons as they will terminate translation when encountered by a ribosome. The table labeled Standard Genetic Code below shows the codon sequence and the corresponding amino‐acid or Stop which it translates to. While this standard genetic code was the first described, as we have sequenced more and more organisms we have found that variant genetic codes exist.

These non‐standard genetic codes come in two varieties. One type may use a particular codon to encode a different amino acid than it does in the standard code. An example of this is C. albicans, where CTG encodes a serine instead of the canonical leucine. The other type is more dramatic; In rare cases, we also see variant genetic codes that encode 21 amino acids, including the twenty canonical amino‐acids and one of the two "non‐standard" amino acids. These are seleno‐cysteine (Sec) and pyrrolysine (Pyl). Each of these non‐standard amino‐acids also has a matching non‐standard tRNA with a special anti‐codon which matches what is normally a Stop codon. Pyrrolysine is encoded by the "amber" stop codon (TAG), one of three codons normally used to signal the termination of mRNA translation. 03‐327/727 2015 [Type text] Pieter Spealman

The use of pyrrolysine has been observed in a small number of microbial species among Archaea, Firmicutes and d‐Proteobacteria possessing the pyrrolysine gene cassette. While these organisms are taxonomically diverse, they possess similar metabolic lifestyles. Each species that uses pyrrolysine is either a methanogen (which produce methane as part of their metabolism) or a methylotroph (which reduce single carbon substrates, such as methane, as part of their metabolism). Pyrrolysine is utilized by these species to perform a specific chemical reaction as part of this metabolism, something which pyrrolysine is especially well suited for. For pyrrolysine to be used in protein synthesis several genes are required. The pyrrolysine biosynthesis genes pylB, pylC, and pylD create the amino‐acid, pyrrolysine, itself. The pylT gene encodes the special pyl‐tRNA whose anti‐codon matches the TAG of the “amber” stop codon. Finally, the pylS gene encodes the aminoacyl‐tRNA synthetase which covalently bonds the amino‐acid and the pyl‐tRNA together. The figure below summarizes the role the amino‐acid biosynthesis enzymes play in pyrrolysine synthesis. PylB uses radical redox chemistry to modify a lysine by inverting the amine group and adding a methyl group to the main chain (blue and green boxes, respectively). PylC uses ATP to drive a condensation reaction covalently bonding together the previously modified lysine and an unmodified lysine (red boxes). PylD catalyzes the oxidation reaction using hydrolysis to replace the terminal amine with a highly reactive aldehyde (yellow boxes). A final spontaneous condensation reaction occurs between the aldehyde and the primary amine allowing for ring formation (purple boxes).

Pyrrolysine Synthesis

03‐327/727 2015 [Type text] Pieter Spealman

Species Tree for Phylogenetics Practica 2015* *Tree constructed by Jayscott Embry (2013)

The above tree shows the full species name, as well as the abbreviation which will be used for the duration of this semester. Each abbreviation is preceded by a letter to help you identify where that species occurs in the greater tree of life. Species preceded by an ‘A’ are Archaea. Species of the bacterial kingdom are divided into delta‐Proteobacteria which are preceded by a ‘D’, and Firmicutes, which are preceded by an ‘F’.

Further information about pyrrolysine, the biosynthetic pathway, and the methane chemistry they are required for can be found in: Functional context, biosynthesis, and genetic encoding of pyrrolysine. Marsha A Gaston, Ruisheng Jiang, and Joseph A Krzycki. Curr Opin Microbiol, 14(3):342–349, Jun 2011. This document is made freely available through NIH here: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3119745/