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 A-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 [14C]pyruvic acid in a [14C]alanine-labeled preparation of purified LPXTGase, which represents 27% of the amino acid composition. Because D-amino 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.
Highlights
Is a characteristic feature of nonribosomally constructed peptide antibiotics (4, 5)
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
As there exists an intriguing possibility that the core protein is 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)
Summary
Growth and [14C]Alanine Labeling of Cells—S. pyogenes, strain D-471, was grown in 1 liter of Todd-Hewitt medium supplemented with 1% yeast extract. The cell pellets were suspended in 1 liter of a synthetic culture medium, and after adding 100 Ci of [14C]alanine Cells were harvested by centrifugation, and the spent medium was saved. A freshly harvested batch of late exponential cells from 1 liter of culture was suspended in the spent medium containing [14C]alanine, and the cells were grown for 1 h and harvested by centrifugation. The combined cell pellets were suspended in 400 ml of 30 mM Mes buffer, pH 6.3, containing 100 mM NaCl, and cells were collected by centrifugation. Purification of [14C]Alanine-labeled LPXTGase—About 3 g of the harvested cell pellets were suspended in 120 ml of 30 mM Mes buffer, pH 6.3. The suspension was centrifuged for 40 min at 10,000 rpm, and the supernatant (membrane extract) was collected.
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