Slow Binding Inhibition of Phospho-N-acetylmuramyl-pentapeptide-translocase (Escherichia coli) by Mureidomycin A

Enzymes of the membrane cycle of reactions in bacterial peptidoglycan biosynthesis remain as unexploited potential targets for antibacterial agents. The first of these enzymes, phospho-N-acetylmuramyl-pentapeptide-translocase (EC 2.7.8.13), has been overexpressed in Escherichia coli and solubilized from particulate fractions. The work of W. A. Weppner and F. C. Neuhaus ((1977) J. Biol. Chem. 252, 2296-2303) has been extended to establish a usable routine fluorescence-based continuous assay for solubilized preparations. This assay has been used in the characterization of the natural product, mureidomycin A as a potent slow binding inhibitor of the enzyme with Ki and Ki∗ of 36 nM and 2 nM, respectively. Enzymes of the membrane cycle of reactions in bacterial peptidoglycan biosynthesis remain as unexploited potential targets for antibacterial agents. The first of these enzymes, phospho-N-acetylmuramyl-pentapeptide-translocase (EC 2.7.8.13), has been overexpressed in Escherichia coli and solubilized from particulate fractions. The work of W. A. Weppner and F. C. Neuhaus ((1977) J. Biol. Chem. 252, 2296-2303) has been extended to establish a usable routine fluorescence-based continuous assay for solubilized preparations. This assay has been used in the characterization of the natural product, mureidomycin A as a potent slow binding inhibitor of the enzyme with Ki and Ki∗ of 36 nM and 2 nM, respectively. INTRODUCTIONEnzymes responsible for the biosynthesis of the peptidoglycan component of the bacterial cell wall are well precedented targets for antibiotics(1.Bugg T.D.H. Walsh C.T. Nat. Prod. Rep. 1992; 9: 199-215Crossref PubMed Scopus (291) Google Scholar). The worldwide emergence of bacterial strains resistant to current antibiotics necessitates the development of new antimicrobial agents(2.Swartz M.N. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 2420-2427Crossref PubMed Scopus (229) Google Scholar, 3.Neu H.C. Science. 1992; 257: 1064-1073Crossref PubMed Scopus (2321) Google Scholar). Phospho-N-acetylmuramyl-pentapeptide-translocase (also translocase I) catalyzes the first step in the membrane cycle of peptidoglycan biosynthesis, namely the transfer of phospho-N-acetylmuramyl-L-Ala-γ-D-Glu-m-DAP1( 1The abbreviations used are: DAPdiaminopimelic aciddansyl5-dimethylaminoanaphthalene-1-sulfonylIPTGisopropyl-1-thio-β-D-galactopyranosideCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic aciddPPdansyl-UDP-MurNAc-pentapeptidekbkilobase pair(s).) -D-Ala-D-Ala from uridine 5′-monophosphate (UMP) to a membrane-bound lipid carrier, undecaprenyl phosphate (see Fig. 1)(4.Struve W.G. Sinha R.K. Neuhaus F.C. Biochemistry. 1966; 5: 82-93Crossref PubMed Scopus (53) Google Scholar, 5.Higashi Y. Strominger J.L. Sweeley C.C. Proc. Natl. Acad. Sci. U.S.A. 1967; 57: 1878-1884Crossref PubMed Scopus (154) Google Scholar). This enzyme is encoded by the mraY gene in Escherichia coli. This gene has been cloned and sequenced; examination of the inferred amino acid sequence indicates that the encoded enzyme is an integral membrane protein whose molecular mass is 39.5 kDa(6.Ikeda M. Wachi M. Jung H.K. Ishino F. Matsuhashi M. J. Bacteriol. 1991; 173: 1021-1026Crossref PubMed Google Scholar). The lipid-linked product of MraY is further elaborated by attachment of an N-acetylglucosamine unit and the precursor is somehow flipped across the membrane and incorporated into peptidoglycan. This and other such lipid-linked cycles have been reviewed(7.Bugg T.D.H. Brandish P.E. FEMS Microbiol. Lett. 1994; 119: 255-262Crossref PubMed Scopus (119) Google Scholar). No commercial antibiotics in current use are directed against translocase I. This enzyme represents a target for novel antibacterial agents which is as yet unexploited.Until recently, the only known inhibitors of this step of peptidoglycan biosynthesis were tunicamycin, which is known also to inhibit mammalian glycoprotein biosynthesis and other lipid-linked glycosyl transfer reactions(8.Tamura G. Sasaki T. Matsuhashi M. Takasaki A. Yamasaki M. Agric. Biol. Chem. 1976; 40: 447-449Crossref Scopus (4) Google Scholar), and amphomycin, which chelates undecaprenyl-P in the presence of Ca2+(9.Tanaka H. Oiwa R. Matsukura S. Omura S. Biochem. Biophys. Res. Commun. 1979; 86: 906-908Crossref Scopus (35) Google Scholar, 10.Banerjee D.K. J. Biol. Chem. 1989; 264: 2024-2028Abstract Full Text PDF PubMed Google Scholar). In recent years two new classes of natural products have been characterized as potent and specific inhibitors of this step in peptidoglycan biosynthesis, the mureidomycins and the liposidomycins(11.Inukai M. Isono F. Takatsuki A. Antimicrob. Agents Chemother. 1993; 37: 980-983Crossref PubMed Scopus (79) Google Scholar, 12.Kimura K. Miyata N. Kawanishi G. Kamio Y. Izaki K. Isono K. Agric. Biol. Chem. 1989; 53: 1811-1815Google Scholar, 13.Ubukata M. Isono K. J. Am. Chem. Soc. 1988; 110: 4416-4417Crossref Scopus (91) Google Scholar, 14.Isono F. Inukai M. Takahashi S. Haneishi T. Kinoshita T. Kuwano H. J. Antibiot. 1989; 42: 667-673Crossref PubMed Scopus (77) Google Scholar). Both classes of compound share a uridine nucleoside moiety found in the substrate UDPMurNAc-pentapeptide, but beyond this there is little obvious similarity to the substrates of translocase I (see Fig. 2). The presence of the common uridine moiety suggests a similar mode of action for this class of molecules.Figure 2:Chemical structures of translocase I substrates and inhibitors.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The mureidomycins are a class of novel peptidylnucleoside antibiotics isolated from Streptomyces flavidovirens SANK 60486 which show selective antipseudomonal activity (minimal inhibitory concentration values of 0.1-3.13 μg/ml), while not being toxic in mice(15.Isono F. Katayama T. Inukai M. Haneishi T. J. Antibiot. 1989; 42: 674-679Crossref PubMed Scopus (65) Google Scholar). Mureidomycin A has been demonstrated to inhibit translocase I activity in particulate preparations from Pseudomonas aeruginosa using a radiochemical assay (IC50, 0.05 μg/ml), but not to significantly inhibit formation of lipid-linked N-acetylglucosamine for teichoic acid synthesis in Bacillus subtilis (IC50 >100 μg/ml) or dolichol-linked precursors for glycoprotein biosynthesis in a mammalian system(11.Inukai M. Isono F. Takatsuki A. Antimicrob. Agents Chemother. 1993; 37: 980-983Crossref PubMed Scopus (79) Google Scholar). The antibacterial potency of mureidomycin A and its novel structure prompted us to begin an investigation into the molecular mechanism of action of this antibiotic. Here we report the characterization of mureidomycin A as a slow-binding inhibitor of solubilized translocase I from Escherichia coli using a convenient fluorescence enhancement continuous assay.EXPERIMENTAL PROCEDURESMaterialsMureidomycin A was isolated as described previously(16.Inukai M. Isono F. Takahashi S. Enokita R. Sakaida Y. Haneishi T. J. Antibiot. 1989; 42: 662-666Crossref PubMed Scopus (67) Google Scholar). Dodecaprenyl phosphate and phosphatidylglycerol were obtained from Sigma. Other chemicals and media were commercially available. Protein was determined using the Smith BCA assay(17.Smith P.K. Krohn R.I. Hermanson G.T. Hallia A.K. Gartner F.H. Provenzano M.D. Fujimoto K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18445) Google Scholar).Construction of Expression Vector pBROC525An 8.6-kb KpnI fragment carrying the mraY gene was subcloned using a DNA preparation made from Kohara phage 110 into the KpnI site of pUC19 to furnish pBROC508(18.Kohara Y. Akiyama K. Isono K. Cell. 1987; 50: 495-508Abstract Full Text PDF PubMed Scopus (1070) Google Scholar). MluI digestion of pBROC508 provided a much smaller 1.3-kb fragment of DNA encoding mraY. MluI overhangs were filled in by treatment with Taq polymerase and the blunt-ended fragment recloned into the SmaI site of pUC19 to give pBROC511, where the direction of transcription of the mraY gene was found to be opposite to that of lacZ. Subcloning the 1.3-kb AflIII/BamHI fragment of pBROC511 into the NcoI/BamHI sites of pTrc99A (Pharmacia Biotech Inc.) gave us pBROC525 where expression of the mraY gene from the trc promoter is regulated by lac Iq. The AflIII restriction site in the mraY gene in pBROC511 is situated directly at the translation start site.Preparation of Phospho-N-acetylmuramyl-pentapeptide-translocaseE. coli JM109 (pBROC525) was grown in LB media at 37°C to OD600nm 1.7, IPTG added to 0.2 mM, and cells harvested after 1.5 h further growth. Cells were opened using an air-driven cell disrupter, whole cells and debris were collected by centrifugation, and membrane fragments were collected by ultracentrifugation (105 × g, 30 min). The pellet was resuspended in 50 mM Tris, pH 7.5, 2 mM β-mercaptoethanol, 1 mM MgCl2, 1 M KCl and stirred for 1 h at 4°C to strip away peripherally associated proteins. Inclusion of MgCl2 in preparative buffers increased the yield of activity 1.5-2-fold. Membrane fragments were collected again, resuspended in buffer without KCl, flash frozen in liquid nitrogen, and stored at −20°C. Enzyme was solubilized from membranes at a protein concentration of 4 mg/ml in 50 mM Tris, pH 7.5, 2 mM β-mercaptoethanol, 1 mM MgCl2, 1% v/v Triton X-100, 20% glycerol with stirring for 1 h at 4°C. Insoluble material was removed by ultracentrifugation (105 × g, 30 min).Preparation of Fluorescent SubstrateUDP-MurNAc-pentapeptide was isolated from cells of B. subtilis W23 based on methodology devised by Lugtenberg et al. (19.Lugtenberg E.J.J. de Haas-Menger L. Ruyters W.H.M. J. Bacteriol. 1972; 109: 326-335Crossref PubMed Google Scholar) and Flouret et al.(20.Flouret B. Mengin-Lecreulx D. van Heijenoort J. Anal. Biochem. 1981; 114: 59-63Crossref PubMed Scopus (63) Google Scholar). UDP-MurNAc-pentapeptide was accumulated in B. subtilis by treatment with chloramphenicol and in cell wall synthesis described (19.Lugtenberg E.J.J. de Haas-Menger L. Ruyters W.H.M. J. Bacteriol. 1972; 109: 326-335Crossref PubMed Google Scholar) and from cells with This preparation was on and only the uridine were using liquid as described B. Mengin-Lecreulx D. van Heijenoort J. Anal. Biochem. 1981; 114: 59-63Crossref PubMed Scopus (63) Google Scholar). was by treatment of the natural substrate with in as described Weppner and Neuhaus Neuhaus F.C. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The compound by on was characterized by and mass was stored as a at for assay is a of the of et H. Oiwa R. Matsukura S. Omura S. Biochem. Biophys. Res. Commun. 1979; 86: 906-908Crossref Scopus (35) Google Scholar). was a from mM Tris, pH 7.5, mM MgCl2, of and UDP-MurNAc-pentapeptide and of particulate protein or of solubilized protein in a In with particulate phosphate was added as a in a of Triton solubilized preparations were was in the assay such that the concentration the Triton in were in a the was removed with a of nitrogen, and the was in the of solubilized enzyme to be were by the of 50 of M pH was with of and in for were made using a fluorescence with an at of phosphate and phosphatidylglycerol were in a and the was removed with a of were in solubilized enzyme as in the radiochemical and to were added in the of assay buffer mM Tris, pH 7.5, mM KCl, 50 mM of of solubilized translocase of and for phosphate and I formation was by of of a concentration of In inhibitor was added with the the the was to to of mM to a in fluorescence in fluorescence with formation of lipid-linked product was at at of coli investigation of the of translocase I activity in coli a specific activity of protein in the of lipid using a radiochemical et M. Wachi M. Jung H.K. Ishino F. Matsuhashi M. J. Bacteriol. 1991; 173: 1021-1026Crossref PubMed Google Scholar) have that expression of the mraY gene in in coli to of translocase I activity in particulate fractions. an pBROC525 was in to the specific of enzyme preparations. This is from pTrc99A and a 1.3-kb DNA fragment the coli mraY gene the of the trc The specific activity of particulate enzyme from coli JM109 (pBROC525) was of protein in the of lipid activity was increased by with IPTG in the with of after in the fluorescence enhancement assay (see JM109 (pBROC525) gave a specific activity of to for JM109 to a of enzyme activity in solubilized preparations by of particulate membrane of coli JM109 and coli JM109 (pBROC525) with IPTG new to the mraY gene product in the of work on translocase I activity in that activity be from membrane preparations with Neuhaus F.C. Biochemistry. PubMed Scopus Google Scholar). A of were for to coli translocase I activity a of using the radiochemical X-100, were found to give of activity after at × these the with the yield of activity was obtained with Triton at a of were used for further preparations of JM109 (pBROC525) solubilized with 1% Triton at 4 of gave specific of to the solubilized enzyme by a of or gave of enzyme No of activity was on that a is being are in to this and Neuhaus have that is a substrate for peptidoglycan synthesis in membrane preparations from S. Neuhaus F.C. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The S. translocase the material with to the natural Neuhaus F.C. J. Biol. Chem. Full Text PDF PubMed Google Scholar). was also demonstrated that this substrate be used for a continuous fluorescence-based assay for translocase I an assay which have not been using radiochemical M. Isono F. Takatsuki A. Antimicrob. Agents Chemother. 1993; 37: 980-983Crossref PubMed Scopus (79) Google Scholar, Neuhaus F.C. J. Biol. Chem. Full Text PDF PubMed Google Scholar, A.K. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The methodology by Neuhaus and has been extended to convenient continuous assay of solubilized translocase I from this assay substrate is into lipid-linked product which the in a in fluorescence of substrate to lipid-linked product in a of the fluorescence at from to and a fluorescence enhancement at of In this assay is of similar to the radiochemical assay the of enzyme to of product in a assay gave a in fluorescence of in a enhancement was found to be directly to protein and at for to 30 not enzyme was to is as in of fluorescence at the yield of the the is not this unit is of with S. translocase I have on undecaprenyl phosphate with the enzyme Neuhaus F.C. Biochemistry. PubMed Scopus Google Scholar) or on of a lipid preparation to enzyme of by with W.G. Neuhaus F.C. Biochemistry. PubMed Scopus Google Scholar). have found that of commercially phosphate or phosphate gave a in in assay the fluorescence enhancement of concentration that the enzyme phosphate and phosphate as of for lipid substrates are the in the are not and undecaprenyl-P is in The concentration of undecaprenyl-P was not into in the of were also with the The in the of added phosphate was of that with phosphate not the to undecaprenyl phosphate is the fluorescence enhancement assay the for has been at and the for was with a of (see Fig. the was at not with a of The and values that is a substrate for translocase I that the enzyme is selective for the substrate which is in to the natural was used in of for for phosphate (see 2). were made using the fluorescence enhancement assay as described in the first concentration with at concentration with at was at values were determined from the using the of the for a Enzyme Large Image Figure ViewerDownload Hi-res image Download of activity in the fluorescence enhancement assay and concentration was (see Fig. The a for mM with activity at mM was by the of 50 mM KCl in the This is with the obtained with the S. R. Neuhaus F.C. Biochemistry. 9: PubMed Scopus Google Scholar). with other that be only by The concentration for was 1 mM, but activity was with the concentration of No activity was using and concentration in the assay was found at Triton of Triton and MgCl2 in the fluorescence enhancement were as that the concentration of of MgCl2 or Triton was Large Image Figure ViewerDownload Hi-res image Download was by of in radiochemical and fluorescence enhancement but not by at the by phosphatidylglycerol is precedented by the of phosphate from a enzyme which catalyzes a similar transfer J. Biol. Chem. 1985; Full Text PDF PubMed Google Scholar). by phosphatidylglycerol and other has also been with a preparation of the S. translocase I activity in 1% Triton X-100, using a radiochemical Neuhaus F.C. J. Biol. Chem. Full Text PDF PubMed Google Scholar). is not to this is to of the of the protein in or of the lipid of I by Mureidomycin with the work of Isono et al. F. K. Inukai M. Antimicrob. Agents Chemother. 1992; PubMed Scopus Google Scholar) with cells of coli mureidomycin A solubilized coli translocase I in the radiochemical assay with the fluorescence enhancement mureidomycin A was found to inhibit translocase I activity in a A of with inhibitor concentration is in Fig. The is characterized by an by a to a mureidomycin A was in of the as described the was not of translocase I by mureidomycin A. in the presence of of mureidomycin A. of fluorescence are is by of slow binding characterized by a in the of inhibitor is to product and to enzyme an fluorescence Large Image Figure ViewerDownload Hi-res image Download (PPT)The is with or slow binding In to and slow binding nM mureidomycin A were to for of and enzyme activity by of mM and in fluorescence to the The of of was of that enzyme is No of potency of the inhibitor was in the that the inhibitor is being the of the the of the be only by slow binding in the presence or of substrates that is not that mureidomycin A with the and were for substrate and in the presence and nM mureidomycin values increased with inhibitor concentration values were that mureidomycin A is with to substrates not In to the we have a mechanism for the enzyme of substrates to after the work of and et W.G. Neuhaus F.C. Biochemistry. PubMed Scopus Google Scholar). have also based on the that mureidomycin A as a inhibitor (see Fig. for the of translocase I by mureidomycin A. and and are enzyme and a W.G. Neuhaus F.C. Biochemistry. PubMed Scopus Google Scholar). is the associated Large Image Figure ViewerDownload Hi-res image Download and for and slow-binding were where is the or for Ki or Ki∗ respectively. and I are the of and mureidomycin respectively. of I values of 36 nM and nM for Ki and (see Fig. In to Ki∗ and values a of inhibitor was to of at enzyme the of the assay a for the of the of at enzyme enzyme an of fluorescence is in the Ki∗ to the Ki to the that Ki∗ is from the much which be with a of the is with the slow binding and a of nM be for of slow-binding of Ki from in the presence of Ki was determined from the as described of Ki∗ from in the presence of Ki∗ was determined from the as described of at enzyme are as a of that in the of and Large Image Figure ViewerDownload Hi-res image Download of the for the to the were determined at inhibitor concentration using Walsh C.T. Biol. 1987; 57: Scholar), where is the at and and are the and The for the of to was determined by against a of inhibitor as in Walsh C.T. Biol. 1987; 57: Scholar). Figure of the for the of to of were a of inhibitor and was determined from the of the of as described Large Image Figure ViewerDownload Hi-res image Download the of this the obtained for was 0.2 whose is with the of the were made to the for the of the by of enzyme from inhibitor by the of 1979; PubMed Scopus Google Scholar). of enzyme was with mureidomycin A the assay described The enzyme was but of activity was is that the mureidomycin with the and is not from the to in the molecular for of translocase I by mureidomycin A we have overexpressed and solubilized the coli enzyme and have a continuous assay for this is not known the of is the well of the trc A was found to inhibit the solubilized activity with nM using a radiochemical the continuous fluorescence enhancement assay we have mureidomycin A as a slow binding enzyme This the first into the molecular mechanism of action of this antibiotic. a of slow binding enzyme inhibitors which other in peptidoglycan biosynthesis such as the of by K. Walsh C.T. Biochemistry. 1988; PubMed Scopus Google Scholar). This mode of enzyme from the of the into a binding In formation of the is to a of the enzyme or in other is to a at the enzyme Walsh C.T. Biol. 1987; 57: Scholar). The that mureidomycin A is with to and that as a inhibitor in the formation of the the of the to the to be are in to the mechanism of translocase I and the of site which also in the mechanism of slow binding by mureidomycin Ki of 36 nM determined for mureidomycin A is a the of the substrate and the Ki∗ a further This binding an for its antibacterial potency in and with an of in mammalian that the of the mechanism of slow binding by mureidomycin A be a for the of novel antibacterial agents. INTRODUCTIONEnzymes responsible for the biosynthesis of the peptidoglycan component of the bacterial cell wall are well precedented targets for antibiotics(1.Bugg T.D.H. Walsh C.T. Nat. Prod. Rep. 1992; 9: 199-215Crossref PubMed Scopus (291) Google Scholar). The worldwide emergence of bacterial strains resistant to current antibiotics necessitates the development of new antimicrobial agents(2.Swartz M.N. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 2420-2427Crossref PubMed Scopus (229) Google Scholar, 3.Neu H.C. Science. 1992; 257: 1064-1073Crossref PubMed Scopus (2321) Google Scholar). Phospho-N-acetylmuramyl-pentapeptide-translocase (also translocase I) catalyzes the first step in the membrane cycle of peptidoglycan biosynthesis, namely the transfer of phospho-N-acetylmuramyl-L-Ala-γ-D-Glu-m-DAP1( 1The abbreviations used are: DAPdiaminopimelic aciddansyl5-dimethylaminoanaphthalene-1-sulfonylIPTGisopropyl-1-thio-β-D-galactopyranosideCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic aciddPPdansyl-UDP-MurNAc-pentapeptidekbkilobase pair(s).) -D-Ala-D-Ala from uridine 5′-monophosphate (UMP) to a membrane-bound lipid carrier, undecaprenyl phosphate (see Fig. 1)(4.Struve W.G. Sinha R.K. Neuhaus F.C. Biochemistry. 1966; 5: 82-93Crossref PubMed Scopus (53) Google Scholar, 5.Higashi Y. Strominger J.L. Sweeley C.C. Proc. Natl. Acad. Sci. U.S.A. 1967; 57: 1878-1884Crossref PubMed Scopus (154) Google Scholar). This enzyme is encoded by the mraY gene in Escherichia coli. This gene has been cloned and sequenced; examination of the inferred amino acid sequence indicates that the encoded enzyme is an integral membrane protein whose molecular mass is 39.5 kDa(6.Ikeda M. Wachi M. Jung H.K. Ishino F. Matsuhashi M. J. Bacteriol. 1991; 173: 1021-1026Crossref PubMed Google Scholar). The lipid-linked product of MraY is further elaborated by attachment of an N-acetylglucosamine unit and the precursor is somehow flipped across the membrane and incorporated into peptidoglycan. This and other such lipid-linked cycles have been reviewed(7.Bugg T.D.H. Brandish P.E. FEMS Microbiol. Lett. 1994; 119: 255-262Crossref PubMed Scopus (119) Google Scholar). No commercial antibiotics in current use are directed against translocase I. This enzyme represents a target for novel antibacterial agents which is as yet unexploited.Until recently, the only known inhibitors of this step of peptidoglycan biosynthesis were tunicamycin, which is known also to inhibit mammalian glycoprotein biosynthesis and other lipid-linked glycosyl transfer reactions(8.Tamura G. Sasaki T. Matsuhashi M. Takasaki A. Yamasaki M. Agric. Biol. Chem. 1976; 40: 447-449Crossref Scopus (4) Google Scholar), and amphomycin, which chelates undecaprenyl-P in the presence of Ca2+(9.Tanaka H. Oiwa R. Matsukura S. Omura S. Biochem. Biophys. Res. Commun. 1979; 86: 906-908Crossref Scopus (35) Google Scholar, 10.Banerjee D.K. J. Biol. Chem. 1989; 264: 2024-2028Abstract Full Text PDF PubMed Google Scholar). In recent years two new classes of natural products have been characterized as potent and specific inhibitors of this step in peptidoglycan biosynthesis, the mureidomycins and the liposidomycins(11.Inukai M. Isono F. Takatsuki A. Antimicrob. Agents Chemother. 1993; 37: 980-983Crossref PubMed Scopus (79) Google Scholar, 12.Kimura K. Miyata N. Kawanishi G. Kamio Y. Izaki K. Isono K. Agric. Biol. Chem. 1989; 53: 1811-1815Google Scholar, 13.Ubukata M. Isono K. J. Am. Chem. Soc. 1988; 110: 4416-4417Crossref Scopus (91) Google Scholar, 14.Isono F. Inukai M. Takahashi S. Haneishi T. Kinoshita T. Kuwano H. J. Antibiot. 1989; 42: 667-673Crossref PubMed Scopus (77) Google Scholar). Both classes of compound share a uridine nucleoside moiety found in the substrate UDPMurNAc-pentapeptide, but beyond this there is little obvious similarity to the substrates of translocase I (see Fig. 2). The presence of the common uridine moiety suggests a similar mode of action for this class of mureidomycins are a class of novel peptidylnucleoside antibiotics isolated from Streptomyces flavidovirens SANK 60486 which show selective antipseudomonal activity (minimal inhibitory concentration values of 0.1-3.13 μg/ml), while not being toxic in mice(15.Isono F. Katayama T. Inukai M. Haneishi T. J. Antibiot. 1989; 42: 674-679Crossref PubMed Scopus (65) Google Scholar). Mureidomycin A has been demonstrated to inhibit translocase I activity in particulate preparations from Pseudomonas aeruginosa using a radiochemical assay (IC50, 0.05 μg/ml), but not to significantly inhibit formation of lipid-linked N-acetylglucosamine for teichoic acid synthesis in Bacillus subtilis (IC50 >100 μg/ml) or dolichol-linked precursors for glycoprotein biosynthesis in a mammalian system(11.Inukai M. Isono F. Takatsuki A. Antimicrob. Agents Chemother. 1993; 37: 980-983Crossref PubMed Scopus (79) Google Scholar). The antibacterial potency of mureidomycin A and its novel structure prompted us to begin an investigation into the molecular mechanism of action of this antibiotic. Here we report the characterization of mureidomycin A as a slow-binding inhibitor of solubilized translocase I from Escherichia coli using a convenient fluorescence enhancement continuous