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Facilitated synthesis of proteins containing modified dipeptides.

ABSTRACT: The elaboration of peptides and proteins containing non-proteinogenic amino acids has been realized using several complementary strategies, including chemical synthesis, ribosome- or non-ribosome-mediated elaboration, intein-mediated polypeptide rearrangements, or some combination of these strategies. All of these have strengths and limitations, and significant efforts have been focused on minimizing the effects of limitations, to improve the overall utility of individual strategies. Our laboratory has studied ribosomally mediated peptide and protein synthesis involving a wide variety of non-proteinogenic amino acids, and in recent years we have described a novel strategy for the selection of modified bacterial ribosomes. These modified ribosomes have enabled the incorporation into peptides and proteins of numerous modified amino acids not accessible using wild-type ribosomes. This has included d-amino acids, β-amino acids, dipeptides and dipeptidomimetic species, as well as phosphorylated amino acids. Presently, we have considered novel strategies for incorporating non-proteinogenic amino acids in improved yields. This has included the incorporation of non-proteinogenic amino acids into contiguous positions, a transformation known to be challenging. We demonstrate the preparation of this type of protein modification by utilizing a suppressor tRNA_CUA activated with a dipeptide consisting of two identical non-proteinogenic amino acids, in the presence of modified ribosomes selected to recognize such dipeptides. Also, we demonstrate that the use of bis-aminoacylated suppressor tRNAs, shown previously to increase protein yields significantly in vitro, can be extended to the use of non-proteinogenic amino acids.

SUBMITTER: Zhang C

PROVIDER: S-EPMC8217302 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Recent papers have reported dipeptides containing d-amino acids to have novel effects that cannot be observed with ll-dipeptides, and such dipeptides are expected to be novel functional compounds for pharmaceuticals and food additives. Although the functions of d-amino acid-containing dipeptides are gaining more attention, there are few reports on the synthetic enzymes that can accept d-amino acids as substrates, and synthetic methods for d-amino acid-containing dipeptides have not yet been constructed. Previously, we developed a chemoenzymatic system for amide synthesis that comprised enzymatic activation and a subsequent nucleophilic substitution reaction. In this study, we demonstrated the application of the system for d-amino acid-containing-dipeptide synthesis. We chose six adenylation domains as targets according to our newly constructed hypothesis, i.e., an adenylation domain located upstream from the epimerization domain may activate d-amino acid as well as l-amino acid. We successfully synthesized over 40 kinds of d-amino acid-containing dipeptides, including ld-, dl-, and dd-dipeptides, using only two adenylation domains, TycA-A from tyrocidine synthetase and BacB2-A from bacitracin synthetase. Furthermore, this study offered the possibility that the epimerization domain could be a clue to the activity of the adenylation domains toward d-amino acid. This paper provides additional information regarding d-amino acid-containing-dipeptide synthesis through the combination of enzymatic adenylation and chemical nucleophilic reaction, and this system will be a useful tool for dipeptide synthesis.IMPORTANCE Because almost all amino acids in nature are l-amino acids, the functioning of d-amino acids has received little attention. Thus, there is little information available on the activity of enzymes toward d-amino acids or synthetic methods for d-amino acid-containing dipeptides. Recently, d-amino acids and d-amino acid-containing peptides have attracted attention as novel functional compounds, and d-amino acid-activating enzymes and synthetic methods are required for the development of the d-amino acid-containing-peptide industry. This study provides additional knowledge regarding d-amino acid-activating enzymes and proposes a unique synthetic method for d-amino acid-containing peptides, including ld-, dl-, and dd-dipeptides.

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Facilitated synthesis of proteins containing modified dipeptides.

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