Interactions of Soluble Penicillin-Binding Protein 2a of Methicillin-Resistant Staphylococcus aureus with Moenomycin

Abstract

Kinetics studies in homogeneous aqueous solution showed that solubilized penicillin-binding protein 2a (sPBP2a) of methicillin-resistant Staphylococcus aureus (a bacterial DD-peptidase) was inhibited by the amphiphilic glycolipid antibiotic moenomycin. Inhibition at the peptidase site was determined by competition experiments between moenomycin and the chromophoric beta-lactam nitrocefin. Under conditions of high salt concentration (1 M NaCl), pseudo-first-order rate constants for the reaction of moenomycin with sPBP2a leading to inhibition of acylation by nitrocefin varied with moenomycin concentration in a biphasic fashion. At low moenomycin concentration (<20 microM) little inhibition occurred, but at higher concentrations a linear increase in rate constant with moenomycin concentration was observed, yielding a second-order rate constant of inhibition of 120 s(-)(1) M(-)(1). Since the cmc of moenomycin under these conditions was shown to be ca. 20 microM, the inhibition was concluded to arise from reaction of sPBP2a with a moenomycin micelle. Protein fluorescence studies showed a pseudo-first-order decrease in fluorescence on reaction of the protein with moenomycin. The variation of this rate constant with moenomycin concentration was consistent with reaction of a moenomycin monomer with the protein with a second-order rate constant of 650 s(-)(1) M(-)(1). This monomer reaction did not occur at the DD-peptidase site since its rate was unaffected by prior acylation of the enzyme by benzylpenicillin; nor did it inhibit reaction at that site by beta-lactams. Under low salt conditions (0.175 M NaCl) where reaction could be studied over a greater range of monomer concentrations since the cmc was ca. 120 microM, similar reactions were involved. Under these circumstances, inhibition was concerted with the reaction of moenomycin monomers, although fast premicellar aggregation of moenomycin with the protein also occurred. All moenomycin interactions with sPBP2a were reversible, as revealed by detergent-extraction chromatography. Lower limits to moenomycin off-rates and equilibrium dissociation constants were 7.7 x 10(-)(4) s(-)(1) and 1.2 microM, respectively. Other amphiphiles did not react in exactly the same manner as moenomycin, indicating some degree of specificity in reactions of the latter. sPBP2a did not have detectable affinity for lipid surfaces (Triton X-114 and phosphatidylglycerol vesicles). A general scheme for reaction of moenomycin with sPBP2a is proposed.

Moenomycin family antibiotics: chemical synthesis, biosynthesis, and biological activity

“…1 is a very stable 181 detergent-like molecule (with a cmc of 0.5 mM at pH 6.8) that tends to aggregate in aqueous solutions and partition into membranes. 88,152,182,183 As a consequence, 1 has a very long halflife in the bloodstream, some hemolytic activity and it is not orally bioavailable. The long lipid chain of 1 is suggested to underlie these undesired effects, 12,96 although this has never been directly tested.…”

An overview of analytical methods for monitoring bacterial transglycosylation

“…While Class B enzymes can only catalyze transpeptidation, Class A enzymes have an N-terminal transglycosylation site as well, making them capable of catalyzing both reactions. 4,14,15 Besides PBPs, monofunctional glycosyl transferases (MGTs), enzymes with non-PBP-related transglycosylation activity, have been discovered in several Gram-positive and -negative organisms. 16 However, because of their high degree of similarity, they are likely to be sensitive to the same inhibitors.…”

Development of a liquid chromatography/mass spectrometry assay for the bacterial transglycosylation reaction through measurement of Lipid II

“…The PBPs can be divided into two classes: class A and class B. Class B enzymes only catalyze transpeptidation, whereas class A enzymes also have an N‐terminal transglycosylation site making them capable of catalyzing both transglycosylation and transpeptidation . In addition, monofunctional glycosyl transferases, enzymes with non‐PBP‐related transglycosylation activity, have been discovered .…”