Rex F. Pratt
The bacterial DD-peptidases or penicillin-binding proteins (PBPs) catalyze the formation and regulation of cross-links in peptidoglycan biosynthesis. β-Lactam antibiotics, which have been studied for many years, restrict bacterial growth by inhibiting these enzymes. PBPs are classified based on their molecular mass into high-molecular mass (HMM) and low-molecular mass (LMM) enzymes, with the latter enzymes subdivided into classes, LMMA, -B, and -C, depending on their function and sequence homologies. The substrate specificity of solubilized PBPs for particular elements of peptidoglycan has not been defined to date.
Previous studies of the substrate specificity of the Streptomyces R61 DD-peptidase (LMMB) revealed that the enzyme shows specificity for the glycyl-L-aminopimelyl segment of its peptidoglycan. Following this strategy, the substrate specificity of LMMC and LMMA PBPs towards specific elements of peptidoglycan of their parent organism was investigated. Small peptidoglycan-mimetic peptides were synthesized and assessed as substrates of LMM PBPs by steady-state kinetics. Peptidoglycan-mimetic peptides containing the D-aminopimelate moiety were efficiently turned over by the LMMC enzymes, which have a well-defined and specific binding site for these peptides. Solubilized LMMA PBPs, on the other hand, were poorly reactive towards small peptidoglycan-mimetic peptides in vitro.
The LMMC PBP, Bacillus subtilis PBP4a, was found to have carboxypeptidase (hydrolyzing terminal D-alanyl-D-alanine) as well as endopeptidase (hydrolyzing cross-linked peptidoglycan) activity from steady-state kinetics studies with peptidoglycan-mimetic peptide substrates. A systematic investigation of the acyl donor and acceptor specificity of B. subtilis PBP4a towards amidated peptidoglycanmimetic peptides was conducted. This enzyme was found to have dual specificity, encompassing both carboxylate and carboxamide containing peptides. The results obtained from molecular modeling studies that were performed starting with the available crystal structures suggested that a neutral histidine residue, His 352, was able to favorably interact with both carboxylate and carboxamide termini of substrates, in agreement with the kinetics data.
The specificity of LMM PBPs for cross-linked pentapeptides in solution was investigated using two synthetic peptidoglycan-mimetic cyclic peptides and steady state kinetics. The larger 19-membered cyclic peptide was found to have higher reactivity than analogous acyclic peptides with two LMMC enzymes (Actinomadura R39 DD-peptidase and Neisseria gonorrhoeae PBP3) and an LMMA PBP (Escherichia coli PBP5) The conformational preference of the cyclic peptides, explored using CD, NMR and molecular modeling experiments, suggested that they are more ordered in solution than the acyclic analogues. The kinetics results were interpreted on the basis of a greater entropic advantage offered by cyclic peptides in bringing about effective catalysis and the enthalpic barrier in achieving the bioactive conformation.
Nemmara, Venkatesh V., "Substrate Specificity of Bacterial DD-Peptidases" (2014). Dissertations. 33.
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