Structure of a lipid intermediate in cell wall peptidoglycan synthesis: a derivative of a C55 isoprenoid alcohol.

Abstract

The peptidoglycan of bacterial cell walls is synthesized in a reaction mechanism in which a membrane-bound lipid serves as a carrier for the sugars and amino acids that eventually become part of the peptidoglycan.' One intermediate in the reaction cycle is represented as lipid-P-P-disaccharide-pentapeptide. This intermediate is common to a number of different bacteria. However, its disaccharidepentapeptide moiety (GlcNAc-MurNAc-L-ala-D-glu-L-lys-D-ala-D-ala)I becomes modified in various ways in different organisms.3 The modified disaccharidepeptide moiety is then transferred to an endogenous acceptor with introduction of a new unit into a growing peptidoglycan.The purpose of the present paper is to report that lipid-P-P-disaccharide-pentapeptide has been obtained in a highly purified form from Micrococcus lysodeikticus, and that the nature of the lipid moiety of this intermediate has been elucidated.Soon after the discovery of the occurrence of a lipid intermediate in peptidoglycan synthesis, a lipid carrier in the biosynthesis of the lipopolysaccharide of the cell wall of Salmonella was described.4' I The structure of this substance is reported in a paper by Wright et al-'Isolation of Lipid-P-P-Disaccharide-Pentapeptide from M. lysodeikticus.-Largescale preparation of the lipid intermediate was carried out using techniques essentially the same as those described previously.7 Particulate enzyme was prepared after lysozyme lysis of cells of M. lysodeikticus8 and incubated with the uridine nucleotide substrates to generate the lipid intermediate. The incubation mixture contained 1000 ml of particulate enzyme prepared in 0.05 M Tris-HCl buffer, pH 7.4, and 0.01 M MgCl2, 1.7 ml of 12 mM UDP-MurNAc-pentapeptide (containing 8.7 X 106 cpm of C14-lysine),9 8 ml of 10 mM UDP-GlcNAc and 30 ml of 5 per cent deoxycholate. Enzyme prepared after lysozyme lysis of cells of M. lysodeikticus is able to synthesize the lipid intermediates, but is unable to utilize these intermediates for peptidoglycan synthesis. Deoxycholate was included in the incubation mixture in order to minimize the addition of endogenous glycine to the lipid intermediate.8After incubation at 200 for 30 minutes, the mixture was extracted with 1 liter of nbutanol:6 M pyridinium acetate (2:1), pH 4.2, followed by two extractions with the same volume of n-butanol. The combined extracts were back-washed with 2 liters of water and then taken to dryness and redissolved in 50 ml of chloroform: methanol (1:1). The yield was 3 X 106 cpm (34%, based on UDP-MurNAc-,C4-pentapeptide).The extract was placed on a column of O-(diethylaminoethyl) cellulose (DEAEcellulose) (2.5 X 25 cm) and eluted as previously described.7 The radioactive eluate obtained with methanol:6 M pyridinium acetate, pH 4.2 (1: 1), was pooled 1878

Dolichol Monophosphate Glucose: An Intermediate in Glucose Transfer in Liver

“…The radioactivity in the lipid increased at first with the time of incubation and decreased subsequently due to its simultaneous disappearance through reaction (2). Therefore accurate measurements of the rate of the glucosylation reaction could not in the smooth and rough microsome fractions showed no great differences in activity but usually more in the former.…”

“…Data on the nature of the products formed by transfer from the glucosylated acceptor lipid (reactions (2) and (3)) are preliminary. One of the few proteins which contain glucose is collagen and transfer from UDPG has been studied.…”

“…The preparations obtained could be used as substrates for reaction (2 After each run the dolichol was extracted from the silica gel with ethyl ether. The purity of the sample was checked by thin-layer chromatography and its identity by its infrared spectrum.…”

Structure of sortase, the transpeptidase that anchors proteins to the cell wall of Staphylococcus aureus

“…The carboxyl group of threonine is amide-linked to the amino group of the pentaglycine crossbridges, thereby anchoring the C-terminal end of surface proteins to the staphylococcal cell-wall peptidoglycan (5,6). It seems that lipid II [undecaprenyl pyrophosphate-MurNAc(-L-Ala-D-iGln-L-Lys(NH 2 -Gly 5 )-D-Ala-D-Ala)-␤1-4-GlcNAc)], a membrane-anchored precursor of cell-wall synthesis (7,8), serves as a substrate for surface protein anchoring (9). Surface proteins tethered to lipid II may subsequently be incorporated into the peptidoglycan by means of the transglycosylation and transpeptidation reactions of bacterial cell-wall synthesis (10).…”

Dolicholmonophosphates: Mannosyl acceptors in a particulate in vitro system of S. cerevisiae

“…As with the endogenous lipid the loss in radioactivity is only observed when GDP and not when GMP is added. This indicates a phosphodiester link in the DMP-mannose analogous to the DMP-glucose described by Behrens and Leloir [9] and the undecaprenol-mannose described by Scher, Lennarz and Sweeley [3] but different from the pyrophosphate bond of other bacterial lipid-intermediates [ 1, 2,4] . The drop in radioactivity of the lipid is very fast; in less than 2 min the back reaction is finished.…”