Construction of Modified Ribosomes for Incorporation of d-Amino Acids into Proteins

Abstract

While numerous biologically active peptides contain D-amino acids, the elaboration of such species is not carried out by ribosomal synthesis. In fact, the bacterial ribosome discriminates strongly against the incorporation of D-amino acids from D-aminoacyl-tRNAs. To permit the incorporation of D-amino acids into proteins using in vitro protein-synthesizing systems, a strategy has been developed to prepare modified ribosomes containing alterations within the peptidyltransferase center and helix 89 of 23S rRNA. S-30 preparations derived from colonies shown to contain ribosomes with altered 23S rRNAs were found to exhibit enhanced tolerance for D-amino acids and to permit the elaboration of proteins containing D-amino acids at predetermined sites. Five specific amino acids in Escherichia coli dihydrofolate reductase and Photinus pyralis luciferase were replaced with D-phenylalanine and D-methionine, and the specific activities of the resulting enzymes were determined.

Origin and Evolution of the Ribosome

“…Thus, a modest peptide of 50 amino acids would perhaps have only two D-amino acids and thus have a good chance of being functional. In fact, incorporation of a D-amino acid into a modern protein is not necessarily destructive to the protein, but instead depends on where it occurs (Dedkova et al 2003(Dedkova et al , 2006.…”

Recognition of Non-α-amino Substrates by Pyrrolysyl-tRNA Synthetase

“…Recently, an altered ribosome, which recognizes Damino acids as well as L-amino acids, was developed. 40,41 Such efforts to engineer ribosome genes will expand the variety of protein backbones by genetic code expansion. The engineering of the 50 S ribosome should facilitate the polymerization of the α-hydroxyacids.…”

Initiating translation with D-amino acids

“…This has been attributed to the fact that either elongation factor (EF-Tu) or ribosome (or possibly both) does not allow D aa-tRNA CUA to read the amber stop codon, resulting in the undesired termination of peptide synthesis executed by release factor. A more recent attempt to use ribosome mutants has given a modest increase in efficiency for the incorporation of (Dedkova et al 2003(Dedkova et al , 2006, but it is yet unclear how generally this approach is applicable to a variety of D aa. Like elongation, the initiation event is also strictly governed by MetRS and initiation factors (IFs) (Kozak 1983;Gold 1988;Gualerzi and Pon 1990).…”