Abstract
Systematic structural modifications of the muramic acid, peptide, and nucleotide moieties of Park’s nucleotide were performed to investigate the substrate specificity of B. subtilis MraY (MraYBS). It was found that the simplest analogue of Park’s nucleotide only bearing the first two amino acids, l-alanine-iso-d-glutamic acid, could function as a MraYBS substrate. Also, the acid group attached to the Cα of iso-d-glutamic acid was found to play an important role for substrate activity. Epimerization of the C4-hydroxyl group of muramic acid and modification at the 5-position of the uracil in Park’s nucleotide were both found to dramatically impair their substrate activity. Unexpectedly, structural modifications on the uracil moiety changed the parent molecule from a substrate to an inhibitor, blocking the MraYBS translocation. One unoptimized inhibitor was found to have a Ki value of 4 ± 1 μM against MraYBS, more potent than tunicamycins.
Highlights
To more thoroughly investigate how structural modification of Park’s nucleotide affects MraY substrate recognition, we first sought to identify a proper polyprenyl phosphate substrate that would be conserved for all the Park’s nucleotide analogues tested
Other polyprenyl phosphates with a shorter length or different configurations still can be recognized as a MraY substrate but their substrate activity is much weaker than undecaprenyl phosphate (C55P) (Supplementary Table 1)
C55P was chosen as the substrate coupling partner for all the Park’s nucleotide analogues studies, and the substrate activity was measured after 1 h reaction for convenient purposes
Summary
To more thoroughly investigate how structural modification of Park’s nucleotide affects MraY substrate recognition, we first sought to identify a proper polyprenyl phosphate substrate that would be conserved for all the Park’s nucleotide analogues tested. Other polyprenyl phosphates with a shorter length or different configurations still can be recognized as a MraY substrate but their substrate activity is much weaker than undecaprenyl phosphate (C55P) (Supplementary Table 1). We describe the systematic preparation of Park’s nucleotides with varying three parts including the peptide, N-substituted muramic acid, and uridine moieties for evaluation as MraYBS substrates (Fig. 2). This information will provide us with the essential moieties and the specificity requirements of the MraY for Park’s nucleotide analogues, as an effort toward development of new inhibitors
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