Biosynthesis of membrane-derived oligosaccharides. Membrane-bound glucosyltransferase system from Escherichia coli requires polyprenyl phosphate

A.C Weissborn,M.K Rumley,E.P Kennedy

doi:10.1016/s0021-9258(18)92940-6

Abstract

The periplasmic glucans of Gram-negative bacteria, including the membrane-derived oligosaccharides (MDO) of Escherichia coli and the cyclic glucans of the Rhizobiaceae, are now recognized to be a family of closely related substances with important functions in osmotic adaptation and cell signaling. The synthesis of the beta-1,2-glucan backbone of MDO is catalyzed by a membrane-bound glucosyltransferase system previously shown to require UDP-glucose and (surprisingly) acyl carrier protein (Therisod, H., Weissborn, A. C., and Kennedy, E. P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7236-7240). In the present study, no glucan intermediates bound to acyl carrier protein or to UDP could detected. The enzyme system, however, was found to be strongly inhibited by bacitracin and by amphomycin. Because the two antibiotics function by forming specific complexes with polyprenyl phosphates, their inhibitory effect suggests a prenol requirement for MDO biosynthesis. Furthermore, the activity of the glucosyltransferase was greatly stimulated by the addition of polyprenyl phosphates such as decaprenyl-P and dihydroheptaprenyl-P, but not by farnesyl-P. The same membrane preparations carry out the synthesis of polyprenyl-P-glucose, which is also stimulated by added polyprenyl-P, including farnesyl-P, the most active of those tested. Pulse chase experiments, however, indicate that the endogenous pool of polyprenyl-P-glucose cannot be an obligate intermediate in the MDO glucosyltransferase system.

Highlights

The periplasmic glucans of Gram-negative bacteria, including the membrane-derived oligosaccharides (MDO) ofEscherichia coliand the cyclic glucans of the Rhizobiaceae, are recognized to be a family of closely related substances with important functions in osmotic adaptationand cell signaling.The synthesis of the j3-1,a-glucanbackbone of MDO is catalyzed by a membrane-bound glucosyltransferase system previously shown to require UDP-glucose and acyl carrier protein
Efficient Conversion of Labeled UDP-glucose to Decaprenyl-P-glucose-When labeled UDP-glucosewas added to the enzyme system at the usual concentration of0.6mM, about 1.5%of the added label was recoveredas decapreny1-Pglucosein the experiment of Table 111.When the same amount of radioactivity was added, but at a much lower concentration of UDP-glucose so that the specific activity was3000-fold higher, 76% of the total count was recovered as decaprenylP-glucose (Table 111).The enzyme catalyzing the synthesis of polyprenyl-P-glucose has a very high affinity for UDP-glucose, as had been indicated by the previous work of Jann et al (1982).This property of the enzyme proved very useful for the pulse-labeling of the endogenous pool of polypreny1-Pglucose in the membrane enzyme preparation
How-Because it was possible that the added p~lyprenyl-P-[~H]ever, the effect of bacitracin and amphomycin in inhibiting glucose did not effectively interact with the membrane-bound the cell-freeglucosyltransferase strongly points to theinvolveenzyme, a pulse-chase experiment was devisedin which a pool ment of somepolyprenylphosphate derivative

Summary

THEJOURNALOF BIOLOGICACLHEMISTRY

0 1991 by The American Society for Biochemistry and Molecular Biology, Inc. Vol 266,No 13, Issue of May 5,pp. 8062-8067,1991 Printed in U.S.A. The periplasmic glucans of Gram-negative bacteria, including the membrane-derived oligosaccharides (MDO) ofEscherichia coliand the cyclic glucans of the Rhizobiaceae, are recognized to be a family of closely related substances with important functions in osmotic adaptationand cell signaling.The synthesis of the j3-1,a-glucanbackbone of MDO is catalyzed by a membrane-bound glucosyltransferase system previously shown to require UDP-glucose and (surprisingly) acyl carrier protein Mutations in the mdoA locus lead to loss of activity of the membrane component of this glucocontrols to which no octyl glucoside was added has been shown to represent the synthesis of labeled /3-1,2-glucan,the backbone structure of MDO (Weissborn and Kennedy, 1984). Pink spots appeared after 5min in an oven at 105 “ C .Purified dihydroheptaprenyl-

MATERIALS AND METHODS

RESULTS

Inhibition of glucosyltransferaseactivity by bacitracin and amphomycin

Findings

DISCUSSION