Phosphoserine Lyase Deoxyribozymes: DNA-Catalyzed Formation of Dehydroalanine Residues in Peptides

Abstract

Dehydroalanine (Dha) is a nonproteinogenic electrophilic amino acid that is a synthetic intermediate or product in the biosynthesis of several bioactive cyclic peptides such as lantibiotics, thiopeptides, and microcystins. Dha also enables labeling of proteins and synthesis of post-translationally modified proteins and their analogues. However, current chemical approaches to introducing Dha into peptides have substantial limitations. Using in vitro selection, here we show that DNA can catalyze Zn2+ or Zn2+/Mn2+-dependent formation of Dha from phosphoserine (pSer), i.e., exhibit pSer lyase activity, a fundamentally new DNA-catalyzed reaction. Two new pSer lyase deoxyribozymes, named Dha-forming deoxyribozymes 1 and 2 (DhaDz1 and DhaDz2), each function with multiple turnover on the model hexapeptide substrate that was used during selection. Using DhaDz1, we generated Dha from pSer within an unrelated linear 13-mer peptide. Subsequent base-promoted intramolecular cyclization of homocysteine into Dha formed a stable cystathionine (thioether) analogue of the complement inhibitor compstatin. These findings establish the fundamental catalytic ability of DNA to eliminate phosphate from pSer to form Dha and suggest that with further development, pSer lyase deoxyribozymes will have broad practical utility for site-specific enzymatic synthesis of Dha from pSer in peptide substrates.

DNA-catalyzed glycosylation using aryl glycoside donors

“…2 More recently, deoxyribozymes have been identified for creation or removal of many common post-translational modifications (PTMs) of peptides, 3 including phosphorylation, 4 dephosphorylation, 5 and formation of dehydroalanine. 6 Glycosylation is an important PTM for which de novo catalysts that allow site-specific peptide and protein modification will have broad utility. 7 Toward the longer-term goal of peptide glycosylation deoxyribozymes, we previously sought DNA-catalyzed glycosylation using the sugar nucleotide UDP-GlcNAc as the glycosyl donor 8 during the key in vitro selection step of each round, followed by capture via NaIO 4 oxidation of the GlcNAc moiety and reductive amination with NH 2 -modified DNA.…”

Catalytic DNA: Scope, Applications, and Biochemistry of Deoxyribozymes

“…Some of this work was recently reviewed [22]; selected highlights are provided here (Figure 3B). Deoxyribozymes have been found for conjugation of oligonucleotides to tyrosine [68–70], tyrosine phosphorylation [71,72], phosphotyrosine (pTyr) and phosphoserine (pSer) dephosphorylation [73], formation of dehydroalanine (Dha) by elimination of phosphate from pSer [74], and lysine modification [75]. The case of tyrosine phosphorylation illustrates that DNA catalysts can distinguish among various peptide substrate sequences [72], which is important for sequence-specific modification.…”

“…In most of these experiments, a crucial aspect was developing the necessary selection methodology, in particular designing a “capture step” that within each selection round is highly selective for the intended reaction product and also sufficiently fast and high-yielding. As one example, identification of Dha-forming deoxyribozymes depends critically upon a thiol-based capture by nucleophilic attack into the electrophilic Dha group, which leads to a PAGE shift solely for those initially very rare DNA sequences that catalyzed formation of Dha (Figure 3C) [74]. …”

“…(B) Examples of DNA-catalyzed peptide side chain and backbone modification [22,68–74]. (C) Capture step during in vitro selection for identification of deoxyribozymes that form dehydroalanine (Dha) from phosphoserine (pSer) [74]. …”

DNAzymes: Selected for Applications

“…Because the removal of a phosphate from a tyrosine only elicits a subtle change in molecular weight between the starting material and the reaction product, they designed an innovative product‐capturing strategy (PCS) to facilitate DNAzyme isolation: another previously selected DNAzyme, named 15MZ36, was used to capture the dephosphorylated product so the desired phosphatase DNAzymes could be isolated by gel electrophoresis. The Silverman group also successfully selected phosphoserine lyase DNAzymes, capable of creating dehydroalanine by eliminating the phosphate group from a phosphoserine that was within a pentapeptide attached to the DNAzyme sequence (Figure B) . On this occasion, they employed a thiol oligonucleotide to capture the reaction product.…”