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

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Abstract
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[N-acetylmuramyl]-peptides were biosynthesized with residues 4 and 5 replaced singly by either D-.alpha.-amino-n-butyric acid [DAbu], D-norvaline or D-valine. The 6 nucleotides were compared with UDP-MurNAc-Ala-DGlu-Lys-DAla-DAla [from Staphylococcus aureus] 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 G. homari and probably requires 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-DGlu-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. The penicillin-sensitive enzyme(s), presumably the transpeptidase, apparently 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.