Presence of D-Alanine in an Endopeptidase from Streptococcus pyogenes

Abstract

D-Amino acids are commonly found in peptide antibiotics and the cell wall peptidoglycan of bacterial cell walls but have not been identified in proteins or enzymes. Here we report the presence of 6 -7 D-alanine residues in an endopeptidase of Streptococcus pyogenes, a unique enzyme involved in surface protein attachment that we term LPXTGase. Using D-amino acid oxidase coupled with catalase for the deamination of D-alanine to pyruvic acid (a conversion unique to D-alanine), we were able to identify [ 14 C]pyruvic acid in a [ 14 C]alaninelabeled preparation of purified LPXTGase, which represents 27% of the amino acid composition. Because Damino acids are not accommodated in ribosomal peptide synthesis, these results suggest that the same process used in assembling peptide antibiotics or a yet unidentified mechanism may synthesize the core protein of this endopeptidase.In our previous report we described the unusual properties of an endopeptidase termed LPXTGase purified from Streptococcus pyogenes (1). This membrane-associated enzyme cleaves a common sequence motif, LPXTG, found near the C termini of precursor proteins programmed to be displayed on the cell surface and linked to the cell wall peptidoglycan. We discovered that the LPXTGase is highly glycosylated and contains hydrophobic amino acids with unusually high masses, which are not among the 20 common amino acids present in ribosomally translated proteins. The presence of unusual amino acids indicated the possibility that the core protein of the enzyme might be constructed entirely or in part through the well characterized nonribosomal peptide synthesis pathway, where amino acids are assembled into a peptide on amino acid-activating multienzyme templates. Examples are the syntheses of peptide antibiotics in Bacilli sp. (2-4), Streptomyces sp. (5-7), and Amycolatopsis sp. (8, 9), synthesis of a phytoionophore in Pseudomonas syringae (10), and synthesis of the immunosuppressant cyclosporin A in the fungus Tolypocladium niveum (11). Unusual amino acids could also be incorporated into the ribosomally translated core protein through post-translational modifications, however. Another unusual feature of the core protein of LPXTGase is that only seven different amino acids represent 93% of the 61 common residues present in the core protein. It is particularly striking that alanine accounts for nearly 40% of the common amino acids found in the protein.Over-representation of one or two amino acids within a peptide is a characteristic feature of nonribosomally constructed peptide antibiotics (4, 5). As there exists an intriguing possibility that the core protein is indeed constructed at least in part by a nonribosomal peptide synthesis pathway, we considered whether some of the alanine residues might be in the D-form, a hallmark feature of nonribosomal synthesis of peptide antibiotics (11,12). This prompted us to search for the presence of D-alanine in the core protein of LPXTGase. In this report, we present strong evidence that alanine in the D-isoform is present in...

Sortase A localizes to distinct foci on the Streptococcus pyogenes membrane

“…In addition to sortase, S. pyogenes harbors a membranelocalized, LPXTG-specific peptidase termed LPXTGase, a unique glycosylated enzyme that contains amino acids in both D and L conformations and noncanonical amino acids (32,33). Studying the relative localization of LPXTGase as compared to sortase may serve as a first step in understanding the relationship between these two LPXTG-specific enzymes.…”

Water Exclusion and Enantioselectivity in Catalysis

“…However, d-a-amino acids are known biomolecules, being found in a number of organisms. [31,32] In any case, using these meteoritic a-amino acids to induce chirality in normal biomolecules is in its infancy, and one can expect much more from it in the future. The other interesting question has to do with amplification.…”

Imitating Prebiotic Homochirality on Earth

“…More important than that, in a random assembly of polypeptides on prebiotic earth (Miller and Orgel 1974), a mixture of L-and D-amino acids would produce a large group of polypeptides with completely random sequences, and therefore a terrible mixture of structures and shapes. (However, some D-amino acids are now seen as very minor components of peptides and even (Lee and Fischetti 2003) of a microbiological protein. ) The ribose in ribonucleic acid is D-ribose, again indicating its 3-dimensional chirality.…”