Regulation of UDP-3-O-[R-3-hydroxymyristoyl]-N-acetylglucosamine Deacetylase in Escherichia coli

Abstract

The first enzyme of lipid A assembly in Escherichia coli is an acyltransferase that attaches an R-3-hydroxymyristoyl moiety to UDP-GlcNAc at the GlcNAc 3-OH. This reaction is reversible and thermodynamically unfavorable. The subsequent deacetylation of the product, UDP-3-O-[R-3-hydroxymyristoyl]-GlcNAc, is therefore the first committed step of lipid A biosynthesis. We now demonstrate that inhibition of either the acyltransferase or the deacetylase in living cells results in a 5-10-fold increase in the specific activity of the deacetylase in extracts prepared from such cells. Five other enzymes of the lipid A pathway are not affected. The elevated specific activity of deacetylase observed in extracts of lipid A-depleted cells is not accompanied by a significant change in the K m for the substrate, but is mainly an effect on V max . Western blots demonstrate that more deacetylase protein is indeed made. However, deacetylase messenger RNA levels are not significantly altered. Inhibition of lipid A biosynthesis must either stimulate the translation of available mRNA or slow the turnover of pre-existing deacetylase. In contrast, inhibition of 3-deoxy-D-manno-octulosonic acid (Kdo) biosynthesis has no effect on deacetylase specific activity. The underacylated lipid A-like disaccharide precursors that accumulate during inhibition of Kdo formation may be sufficient to exert normal feedback control.

Lipid A Modification Systems in Gram-Negative Bacteria

“…LpxC levels increase five-to tenfold in cells treated with sublethal doses of LpxC inhibitors (109). Induction is not associated with increased transcription (109) but may be due to reduced LpxC turnover when the early steps of Kdo 2 -lipid A biosynthesis are curtailed.…”

UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine Deacetylase Functions through a General Acid-Base Catalyst Pair Mechanism

“…LpxC Assay-The deacetylase activity was measured as described previously (6,23). Briefly, assay mixtures containing buffer, bovine serum albumin (fatty acid-free, 1 mg/ml), triscarboxyethylphosphine (0.5 mM), and […”

Effect of Cold Shock on Lipid A Biosynthesis inEscherichia coli

“…The deacetylase (LpxC) that catalyzes the second, committed step of lipid A biosynthesis is increased about 10-fold in cells treated with the specific deacetylase inhibitor, L-573,655 (44,45), or in point mutants with low levels of lpxA (44,46). Deacetylase regulation is not accompanied by significant changes in lpxC mRNA (44). Instead, LpxC protein levels may be controlled by the membrane-associated protease FtsH (47).…”