Steric effects on penicillin-sensitive peptidoglycan synthesis in a membrane-wall system Gaffkya homari

Residues 4 and 5 of the pentapeptide moiety, R-Ala1-DGlu2-Lys3-DAla4-DAla5, of peptidoglycan play an important role in the donor phase of cross-linked glycan synthesis. To assess the role of these residues in this phase, a series of UDP-MurNAc-peptides were biosynthesized with residues 4 and 5 replaced singly by either D-alpha-amino-n-butyric acid, D-norvaline, or D-valine. The six nucleotides were compared with UDP-MurNAc-Ala-DGlu-Lys-DAla-DAla (reference) in nascent (penicillin-insensitive) peptidoglycan synthesis and in penicillin-sensitive peptidoglycan synthesis. The synthesis of penicillin-sensitive peptidoglycan is catalyzed by membrane-walls isolated from Gaffkya homari and would appear to require the concerted action of transglycosylase and transpeptidase. The membrane-wall system shows a high degree of discrimination for the steric substituents, -CH3 and -CH2CH3, in residue 4. For example, for UDP-MurNAc-Ala-DGly-Lys-DAbu-DAla and -Ala-DGlu-Lys-DAla-DAbu, Vmax/km is 0.19 and 0.95 and Vmax is 0.03 and 0.52, respectively, of the value for the reference nucleotide. In contrast, for the synthesis of nascent peptidoglycan with these nucleotides Vmax/Km is 0.75 and 0.80, and Vmax is 0.71 and 1.0, respectively, of the value for the reference nucleotide. This trend was also illustrated with the other nucleotides in the time course experiments. These results indicate that the penicillin-sensitive enzyme(s), presumably the transpeptidase, has a higher degree of specificity in the donor phase for D-alanine in residue 4 than for D-alanine in residue 5 in the cross-linking stage of peptidoglycan synthesis.