Didier Blanot

A MurG assay which utilises a synthetic analogue of lipid I

A standard assay for the MurG enzyme using a lipid I analogue [MurNAc(N(epsilon)-dansylpentapeptide)-pyrophosphoryl (R,S)-alpha-dihydroheptaprenol] and radioactive UDP-N-acetylglucosamine was set up. A high concentration (35%) of dimethylsulfoxide was necessary for maximal activity. Separation and quantitation were accomplished by reverse-phase high performance liquid chromatography (HPLC) in isocratic conditions and on-line radioactivity detection, thereby providing a rapid and accurate assay. The kinetic parameters of the MurG reaction were determined; the reaction was shown to also catalyse the reverse reaction at a measurable rate. A lipid I analogue containing dihydroundecaprenol as the prenyl chain turned out to be a poor MurG substrate, presumably owing to aggregation.

Formation of adenosine 5′‐tetraphosphate from the acyl phosphate intermediate: a difference between the MurC and MurD synthetases of Escherichia coli

The mechanism of the Mur synthetases of peptidoglycan biosynthesis is thought to involve in each case the successive formation of an acyl phosphate and a tetrahedral intermediate. The existence of the acyl phosphates for the MurC and MurD enzymes from Escherichia coli was firmly established by their in situ reduction by sodium borohydride followed by acid hydrolysis, yielding the corresponding amino alcohols. Furthermore, it was found that MurD, but not MurC, catalyses the synthesis of adenosine 5′‐tetraphosphate from the acyl phosphate, thereby substantiating its existence and pointing out a difference between the two enzymes.

Synthesis of an analogue of the lipoglycopeptide membrane intermediate I of peptidoglycan biosynthesis

α-Dihydroheptaprenyl-pyrophosphoryl-N-acetylmuramoyl-L-Ala-γ-D-Glu-meso-diaminopimeloyl(N ∈-dansyl)-D-Ala-D-Ala (1), an analogue of lipid I of peptidoglycan biosynthesis, was synthesized from natural UDP-N-acetylmuramoyl-pentapeptide in three steps. Compound1 was shown to be a substrate for the MurG transferase fromEscherichia coli, even in the absence of membranes. When membranes were present, dansylated peptidoglycan was also formed.

Effect of analogues of diaminopimelic acid on the meso-diaminopimelate-adding enzyme from Escherichia coli.

Several analogues of diaminopimelic acid (A2pm) were tested as substrates or inhibitors of the meso‐diaminopimelate‐adding enzyme from Escherichia coli. They included lanthionine derivatives, a phosphonic analogue, heterocyclic compounds, 3‐fluoro‐A2pm, 4‐methylene‐A2pm and N‐hydroxy‐A2pm. The best substrates were, in decreasing order of specific enzyme activity, (2S,3R,6S)‐3‐fluoro‐A2pm, meso‐lanthionine sulfoxide and N‐hydroxy‐A2pm (mixture of stereoisomers). In those cases where all the stereoisomers were available, the specificity could be described as meso ⪢ DD ≈ LL. N‐Hydroxy‐A2pm (mixture of stereoisomers) strongly inhibited the addition of radioactive meso‐A2pm to UDP‐N‐acetylmuramoyl‐dipeptide.

Synthesis of α and β anomers of UDP-N-acetylmuramic acid☆

UDP-N-acetylmuramyl (UDP-MurNAc) derivatives are substrates for several cytoplasmics steps of the synthesis of bacterial peptidoglycan (Park, 1952). Their availability is a prerequisite for developing the detailed study of the synthetases catalyzing these reactions. Since they are not commercial compounds, they have to be prepared from bacterial cells in which they accumulate under specific conditions. However, such procedures are long and tedious, and yields are often low. An alternative approach is to chemically synthesize UDP-N-acetylmuramic acid on a large scale, and to use it as starting material for the in vitro enzymatic preparation of the other UDP-MurNAc precursors. In this communication, we wish to report the total synthesis of UDP-MurNAc.

Synthesis of new peptide inhibitors of the meso-diaminopimelate-adding enzyme

Abstract In order to obtain inhibitors of the meso-diaminopimelate-adding enzyme, which catalyzes an early reaction in the biosynthesis of bacterial peptidoglycan, several new peptide derivatives of general formula Nα-propionyl- l -alanyl- d (or ambo)-Xaa were synthesized: four transition-state analogs (Xaa = phosphinothricin, homocysteic acid, buthionine sulfoximine, buthionine sulfoximine phosphate), three analogs of γ- d -glutamyl phosphate (Xaa = 3-phosphonoacetamido-alanine, 3-phosphonomethylaminoaspartic acid, 2-amino-6-phosphonohexanoic acid), and one derivative with Xaa = ornithine. After preincubation with partially purified meso-diaminopimelate-adding enzyme from Escherichia coli, peptide derivatives with Xaa = buthionine sulfoximine, 3-phosphonoacetamido-alanine and 2-amino-6-phosphonohexanoic acid appeared to be among the best inhibitors obtained up to date, with I50 values between 0.6 and 1.2 mM. When the two first compounds were tested for in vitro antibacterial activity, they gave negative results.