D-Amino Acid Probes for Penicillin Binding Protein-based Bacterial Surface Labeling [Enzymology]

October 23rd, 2015 by Fura, J. M., Kearns, D., Pires, M. M.

Peptidoglycan is an essential and highly conserved mesh structure that surrounds bacterial cells. It plays a critical role in retaining a defined cell shape and, in the case of pathogenic Gram-positive bacteria, it lies at the interface between bacterial cells and the host organisms. Intriguingly, bacteria can metabolically incorporate unnatural D-amino acids into the peptidoglycan stem peptide directly from the surrounding media, a process mediated by penicillin binding proteins (PBPs). Metabolic peptidoglycan remodeling via unnatural D-amino acids has provided unique insight into peptidoglycan biosynthesis of live bacteria and has also served as the basis of a synthetic immunology strategy with potential therapeutic implications. A striking feature of this process is the vast promiscuity displayed by PBPs in tolerating entirely unnatural sidechains. Yet, the chemical space and physical features of this sidechain promiscuity has not been systematically determined. In this report, we designed and synthesized a library of variants displaying diverse sidechains to comprehensively establish the tolerability of unnatural D-amino acids by PBPs in both Gram-positive and Gram-negative organisms. In addition, nine Bacillus subtilis PBP-null mutants were evaluated with the goal of identifying a potential primary PBP responsible for unnatural D-amino acid incorporation and gaining insight into temporal control of PBP activity. Together, we have empirically established the scope of physical parameters that govern metabolic incorporation of unnatural D-amino acids into bacterial peptidoglycan.