Methanosarcina spp. are capable of utilizing methylamines as growth substrates for methanogenesis. The methylamine methyltransferases responsible for initiating this process contain an unusual amino acid, pyrrolysine, encoded by an in-frame amber codon. This dissertation examines how pyrrolysine has infiltrated the genetic code of Methanosarcina acetivorans, and the rationale for this amino acid being maintained.
Amber translation as pyrrolysine requires a specific pyrrolysyl-tRNA synthetase, encoded by pylS, that catalyzes the ligation of pyrrolysine onto an amber-decoding tRNA (tRNApyl), encoded by pylT. Expression of the pylTand pylS genes in E. coli, in the presence of an exogenous source of pyrrolysine, is sufficient to allow amber translation as pyrrolysine in this organism (Chapter 2).
In the Methanosarcina spp., three other genes, pylB, pylC, pylD, have been identified that are thought to be co-transcribed with pylTS. Co-expression of pylBCD with pylTand pylS in E. coli allowed amber translation as pyrrolysine to occur in the absence of an exogenous source of pyrrolysine, showing that PylB, PylC and PylD play a role in pyrrolysine biosynthesis. This suggests the pyl operon acts as a genetic code expansion cassette allowing the transmissible genetic encoding of pyrrolysine (Chapter 3).
A major question for amber translation as pyrrolysine was whether a cis-acting element, dubbed the pyrrolysine insertion sequence PYLIS, was required. Recombinant expression of the monomethylamine methyltransferase gene, mtmB, in the native organism Methanosarcina acetivorans allowed this problem to be addressed. In the absence of PYLIS, amber-translation as pyrrolysine was still observed generating approximately 30% of the full-length product compared to wild-type levels. Whilst showing PYLIS is not essential of UAG translation, the decrease in production of fulllength methylamine methyltransferases would be problematic for the methanogen, which produces little energy from methanogenesis (Chapter 4).
This system has enabled functional studies to be performed on MtmB by allowing the replacement of pyrrolysine, and residues within hydrogen-bonding distance of pyrrolysine, with other amino acids by site-directed mutagenesis. These replacements lead to a loss of MtmB activity, whilst maintaining the ability to bind the cognate corrinoid protein, MtmC. Combined, this suggests that pyrrolysine is essential for methanogenesis from methylamines (Chapter 5).