Enzymes for biosynthesis and utilization of the 22nd genetically encoded amino acid, pyrrolysine. Crystal structures, reaction mechanisms and applications in biotechnology

• Applicant: Professor Dr. Michael Groll
• Subject Area: Biochemistry
• Term: from 2012 to 2015
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 226560260

Pyrrolysine was discovered in 2002 as the 22nd genetically encoded amino acid. Specifically, the tRNA and the cognate aminoacyl tRNA synthetase, derived from the pylT and pylS-genes, jointly enable the read-through of UAG stop codons by the protein biosynthetic machinery under insertion of the unusual amino acid into protein sequences (Hao et al. 2002; Srinivasan et al. 2002). Whereas pyrrolysine is so far only known to be incorporated into few proteins involved in the methane metabolism in the family Methanosarcinaceae and a few eubacteria, the pyrrolysine biochemistry has been rapidly recognized as a fascinating tool for introducing unusual amino acids into proteins. Pyrrolysine has been shown to be synthesized by the consecutive action of three proteins specified by the pylB, pylC and pylD genes (Gaston et al. 2011). The biosynthetic pathway has been deduced from compelling indirect evidence, but none of the three proteins had been characterized structurally and functionally. We have determined the X-ray structure of the S-adenoslymethionine iron sulfur protein PylB (Quitterer et al., 2012; see 2.3) and aim to use these data as a starting point for investigating the mechanism and the regulation of PylB protein. In this research project we would like to clone and express PylC and PylD protein as well, determine their structures by X-ray crystallography and investigate their reaction mechanisms. In addition, our goal is to prepare pyrrolysine and pyrrolysine derivatives in large quantities by using either PylBCD proteins or chemical synthesis. Next, we will establish a modified expression system in E. coli that, when exposed to the distinct amino acids and transformed with a polynucleotide comprising an in-frame UAG codon, inserts these residues into the target protein at defined positions. Hereby, our focus will be to functionally and structurally analyze proteins exhibiting pyrrolysine or pyrrolysine derivatives in their catalytic active sites.

• DFG Programme: Research Grants