Exploring the Active Site of the Antibacterial Target MraY by Modified Tunicamycins

Abstract

The alarming growth of antibiotic resistance that is currently ongoing is a serious threat to human health. One of the most promising novel antibiotic targets is MraY (phospho-MurNAc-pentapeptide-transferase), an essential enzyme in bacterial cell wall synthesis. Through recent advances in biochemical research, there is now structural information available for MraY, and for its human homologue GPT (GlcNAc-1-P-transferase), that opens up exciting possibilities for structure-based drug design. The antibiotic compound tunicamycin is a natural product inhibitor of MraY that is also toxic to eukaryotes through its binding to GPT. In this work, we have used tunicamycin and modified versions of tunicamycin as tool compounds to explore the active site of MraY and to gain further insight into what determines inhibitor potency. We have investigated tunicamycin variants where the following motifs have been modified: the length and branching of the tunicamycin fatty acyl chain, the saturation of the fatty acyl chain, the 6″hydroxyl group of the GlcNAc ring, and the ring structure of the uracil motif. The compounds are analyzed in terms of how potently they bind to MraY, inhibit the activity of the enzyme, and affect the protein thermal stability. Finally, we rationalize these results in the context of the protein structures of MraY and GPT.

Branched Chain Lipid Metabolism As a Determinant of the N-Acyl Variation of Streptomyces Natural Products

“…Similarly, exogenous straight chain fatty acids (palmitate, oleate) or a mixture of metabolically deuterated BCFAs isolated from B. subtilis were not utilized for TUN biosynthesis during fermentation of S. chartreusis. Somewhat more surprisingly, neither exogenous supplied [1-13 C]acetate nor [2- 13 C]acetate were incorporated into TUN. Moreover, sublethal concentrations of known inhibitors of BCAA or BCFA metabolism, including triclosan and triclocarban (targeting bacterial enoyl-acyl carrier protein reductase enzyme, FabI), cerulenin (targeting bacterial βketoacyl−acyl carrier protein synthases), and isoniazid (targeting mycolic acid enoyl-acyl carrier protein reductase, InhA) had no observable effect on the TUN profiles produced by either S. chartreusis NRRL 12338 or NRRL 3882.…”

“…17,18 Moreover, varying either fatty acyl chain length or branching type has a significant effect on inhibition of bacterial MraY from C. bolteae. 13 This may be due in part to a Phe residue (F162 in CbMraY) that restricts the tunnel-shaped cavity where the TUN fatty acyl chain binds. 13 Interestingly, a quite different response to chain length variation is seen in eukaryotic GPT, 56 which may be related to the fact that the corresponding Phe group is replaced by a smaller Leu residue.…”

“…Originally only iso-type TUNs were reported, 12 but more recently anteiso-type TUNs have been found due to improved chromatography techniques. 13 Irrespective of the branching type, the Streptomyces-produced TUN (and also QVM and STV) contain an unusual conjugated trans-2,3double bond (Figure 1), although smaller amounts of saturated variants are also found. 3 The Rathayibacter-produced CORs are more diverse and also contain several 3-hydroxyacyl groups, with both iso-and anteiso-branched chains (Supporting Information Figure S1).…”

“…The N -acyl groups on the TUN family of natural products are generally medium sized C13–C19, odd and even chain lengths, and can be either branched or unbranched ,, (Figure ). Originally only iso -type TUNs were reported, but more recently anteiso -type TUNs have been found due to improved chromatography techniques .…”

“…The N -acyl groups on the TUN family of natural products are generally medium sized C13–C19, odd and even chain lengths, and can be either branched or unbranched ,, (Figure ). Originally only iso -type TUNs were reported, but more recently anteiso -type TUNs have been found due to improved chromatography techniques . Irrespective of the branching type, the Streptomyces -produced TUN (and also QVM and STV) contain an unusual conjugated trans -2,3-double bond (Figure ), although smaller amounts of saturated variants are also found .…”