New Insight into the Catalytic Mechanism of Bacterial MraY from Enzyme Kinetics and Docking Studies

Abstract

Phospho-MurNAc-pentapeptide translocase (MraY) catalyzes the synthesis of Lipid I, a bacterial peptidoglycan precursor. As such, MraY is essential for bacterial survival and therefore is an ideal target for developing novel antibiotics. However, the understanding of its catalytic mechanism, despite the recently determined crystal structure, remains limited. In the present study, the kinetic properties of Bacillus subtilis MraY (BsMraY) were investigated by fluorescence enhancement using dansylated UDP-MurNAc-pentapeptide and heptaprenyl phosphate (C35-P, shortchain homolog of undecaprenyl phosphate, the endogenous substrate of MraY) as second substrate. Varying the concentrations of both of these substrates and fitting the kinetics data to two-substrate models showed that the concomitant binding of both UDPMurNAc-pentapeptide-DNS and C35-P to the enzyme is required before the release of the two products, Lipid I and UMP. We built a model of BsMraY and performed docking studies with the substrate C35-P to further deepen our understanding of how MraY accommodates this lipid substrate. Based on these modeling studies, a novel catalytic role was put forward for a fully conserved histidine residue in MraY (His-289 in BsMraY), which has been experimentally confirmed to be essential for MraY activity. Using the current model of BsMraY, we propose that a small conformational change is necessary to relocate the His-289 residue, such that the translocase reaction can proceed via a nucleophilic attack of the phosphate moiety of C35-P on bound UDP-MurNAc-pentapeptide.Among the enzymes involved in bacterial peptidoglycan synthesis, phospho-N-acetylmuramyl-pentapeptide translocase (MraY 4 ; EC 2.7.8.13) has been studied extensively (1, 2). This enzyme performs the initial membrane step in this process, forming undecaprenyl-N-acetylmuramyl-pentapeptide (Lipid I) from UDP-N-acetylmuramyl-pentapeptide (UDP-MurNAcpentapeptide) and undecaprenyl phosphate, in both Gram-positive and Gram-negative bacteria. Given the role of MraY in bacterial cell wall synthesis (3, 4) and cell growth (5), this enzyme is an interesting target for antibacterial drugs. Recently, the crystal structure of MraY from the Gram-negative species Aquifex aeolicus (Protein Data Bank entry 4J72) was determined (6). The enzyme was extracted from its membrane environment with detergent and crystallized as a symmetrical homodimer. Each protomer consists of 10 transmembrane helices, with both the N and C termini locating on the periplasmic side (outside) of the cytoplasmic membrane (1). Before the publication of this high resolution (3.3 Å) structure, other studies attempted to unravel the catalytic mechanism of action of MraY by site-directed mutagenesis and kinetics studies, using either membrane-embedded MraY or detergent-extracted and purified preparations (1,7,8). These studies proposed that catalysis proceeds most likely via a one-step process, although a two-step process has also been suggested (1). In the single-step process, a ternary complex of MraY, UDP-M...

The Membrane Steps of Bacterial Cell Wall Synthesis as Antibiotic Targets

“…It was found that increase of C55-P concentration increased the sensitivity of the assay to inhibition by tunicamycin. This finding corresponds to the Km discrepancies of MraY between different studies when different C55-P concentrations were used, which is addressed as the major message in our own study [ 34 ]. This FRET assay allowed the measurement of the fluorescence intensity ratio of the donor and acceptor, hence fluctuations in measurements that influence both the donor and acceptor fluorescence could be ruled out.…”

“…It is worth noting that in this study, the kinetic values of BsMraY and its mutants were obtained by varying UDP-Mpp concentration between 3 and 6 mM while keeping C55-P at 1.1 mM. These concentrations are very high, and the K M values obtained were much higher than those of our own kinetic study [ 34 ]. The K cat value of the H289R mutant reported in this study decreased by three orders of magnitude.…”

“…However, the two studies used different concentrations of the lipid carrier C55-P in the synthesis reaction, namely 1.1 mM in [ 32 ] and 50 µM in [ 33 ], suggesting that the true K M value of BsMraY for UDP-Mpp is different from these apparent K M values. Indeed, more recent kinetics studies on BsMraY showed that the apparent affinity of either substrate to the enzyme is dependent on the concentration of the other [ 34 , 35 ]. This characteristic has been overlooked by the other published studies.…”

“…The lack of structural information at the time limited the understanding of the mechanism of MraY, which made the reasons for the dependence on polar lipids unclear. Recent observations [ 34 , 35 ] indicate that an exchange reaction between UMP and UDP-Mpp does not occur unless C55-P is present, and the previously-suggested covalent enzyme-substrate intermediate, MraY-phospho-MurNAc-pp, is questionable [ 41 ]. Lloyd et al [ 41 ] claimed that an intermediate was trapped in the reaction mixture without the addition of C55-P, an E. coli membrane preparation was used instead of pure MraY enzyme; therefore, the presence of a small amount of C55-P cannot be excluded.…”

“…It was previously reported that mureidomycin A is competitive with both UDP-Mpp and C55-P, as the Km values of MraY for both substrates altered upon changing the inhibitor concentration [ 31 ]. This conclusion should be re-visited since the K M value of either substrate is directly influenced by changing the concentration of the other [ 34 ]. This suggests that the inhibition of one substrate could indirectly influence the Km value for the other substrate without competitively inhibiting it.…”