Biosynthesis of a water-soluble lipid I analogue and a convenient assay for translocase I

Abstract

Translocase I (MraY/MurX) is an essential enzyme in growth of the vast majority of bacteria that catalyzes the transformation from UDP-MurNAc-pentapeptide (Park’s nucleotide) to prenyl-MurNAc-pentapeptide (lipid I), the first membrane-anchored peptidoglycan precursor. MurX has been received considerable attentions to the development of new TB drugs due to the fact that the MurX inhibitors kill exponentially growing Mycobacterium tuberculosis (Mtb) much faster than clinically used TB drugs. Lipid I isolated from Mtb contains the C50-prenyl unit that shows very poor water-solubility, and thus, this chemical characteristic of lipid I renders MurX enzyme assays impractical for screening and lacks reproducibility of the enzyme assays. We have established a scalable chemical synthesis of Park’s nucleotide-Nε-dansylthiourea 2 that can be used as a MurX enzymatic substrate to form lipid I analogues. In our investigation of minimum structure requirement of the prenyl phosphate in the MraY/MurX-catalyzed lipid I analogue synthesis with 2, we found that neryl phosphate (C10-phosphate) can be recognized by MraY/MurX to generate the water-soluble lipid I analogue in quantitative yield under the optimized conditions. Herein, we report a rapid and robust analytical method for quantifying MraY/MurX inhibitory activity of library molecules.

Fluorescence-based assay for polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA) and identification of novel antimycobacterial WecA inhibitors

“…MurX assay was performed via the procedure reported previously (31). Park’s nucleotide- N ε -C 6 -dansyl and neryl phosphate were chemically synthesized according to the reported procedures (22–24).…”

Solid-Phase Modular Synthesis of Park Nucleotide and Lipids I and II Analogues

“…In addition to the fact that the analogues of Park nucleotide, lipids I and II, which are precursors to peptidoglycan, have the potential to be inhibitors of peptidoglycan biosynthesis, 5,6) they are used as biological tools to elucidate the biology of bacterial cell walls. For example, dansylated Park nucleotide has been used as a fluorescent substrate in an assay screening of MraY inhibitor, 7) and lipids I and II analogues with short lipid tails, such as a neryl group instead of a undecaprenyl group, have been used in mechanistic studies of MurY and MurG 8,9) (Fig. 2, 2 and 3).…”

Chemoenzymatic syntheses of water-soluble lipid I fluorescent probes

“…Thus, the machinery for PG synthesis is still considered to be a source of unexploited drug targets. 4,5 We have been studying bacterial phosphotransferases and glycosyltransferases aiming at developing new antibacterial agents for multi-drug resistant (MDR) pathogens. 5–13 Especially, the membrane-associated proteins translocase I (MraY/MurX) and MurG, which catalyze the transformations from Park’s nucleotide (UDP-MurNAc-pentapeptide) to lipid I (MurNAc(pentapeptide)-pyrophosphoryl prenol) and from lipid I to lipid II (GlcNAc-MurNAc(pentapeptide)-pyrophosphoryl prenol), are our research interests (Fig.…”

“…5 A water-soluble lipid I generated in the reaction could be quantitated conveniently via reverse-phase HPLC without tedious extraction procedures. These synthetic protocols could be applied to a development of robust MraY/MurX assay for identifying novel antibacterial agents.…”

“…29 When Hy MraY was applied to the transformation from Park’s nucleotide- N [ε] -dansylthiourea 1 to neryl-lipid I- N [ε] -dansylthiourea 7 with neryl phosphate (60 equivalents), denaturing of the purified MraY was observed during long reaction time required for neryl-lipid I synthesis; the reaction was terminated in 40–60% conversion within 1h, whereas, the same reaction with M. smegmatis P-60 furnished the desired product 7 in near quantitative yield after 12h (in 0.075 mM solution for Park’s nucleotide). 5 Using membrane fractions (e.g. P-60) are convenient approach for pilot scale syntheses and especially for biological assays.…”