Structure of a lipid intermediate in cell wall peptidoglycan synthesis: a derivative of a C55 isoprenoid alcohol.
Year: 1967
Type: article
Abstract: The peptidoglycani 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.1 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-M\/urNAc-L-ala-D-glu-L-lys-D-ala-D-ala)2 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 Mlicrococcus lysodeikticus, and that the nature of the lipid moiety of this intermediate has beeln elucidated. Soon after the discovery of the occurrence of a lipid intermediate in peptidoglyean synthesis, a lipid carrier in the biosynthesis of the lipopolysaccharide of the cell wall of Salmonella was described.4' The structure of this substance is reported in a paper by Wright et al.6 Isolation of Lipid-P-P-Disaccharide-Pentapeptide fromn M. iysodeikticus. 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 111. lysodeilticus8 and incubated with the uridine nucleotide substrates to generate the lipid intermediate. The incubation mixture contained 1000 ml of particulate enzyme prepared in 0.05 111 Tris-HCl buffer, pH 7.4, and 0.01 M M\4gCl2, 1.7 ml of 12 mM\4 UDP-1\'furNAc-pentapeptide (containing 8.7 X 106 cpm of C'4-lysine),9 8 ml of 10 mM\4 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 peptidoglyean synthesis. Deoxycholate was included in the incubation mixture in order to minimize the addition of endogenous glycine to the lipid intermediate.8 After incubation at 20? 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-M\'SurNAc-C14pentapeptide). 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
Authors: Y Higashi, J L Strominger, C C Sweeley
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2010589798