No 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.

No abstract

Bacterial peptidoglycan biosynthesis includes four enzymatic reactions in which successive amino acid residues are ligated to uridine diphospho-N-acetylmuramic acid (UDP-MurNAc). By comparing the amino acid sequences of MurC, -D, -E, and -F proteins from various bacterial genera, four regions of homology were identified. A profile search of Swissprot for related sequences revealed that these regional similarities were present in the folyl-gamma-polyglutamate ligases. These sequence homologies appear to track with catalytic function: both enzyme families proceed through an ordered kinetic mechanism and form product via an acyl phosphate intermediate. Two highly conserved residues in region II were examined through site-directed mutagenesis of the murein D-alanyl-D-alanine-adding enzyme from Escherichia coli (murF; E158 and H188). All mutations were highly detrimental to activity with enzyme specific activity reductions of 200-4500-fold, validating the critical nature of these residues. DNA sequence analysis from three E. coli mutants harboring the murC3 (G344D), murE1 (G344K, A495S), and murF2 (A288T) mutations revealed the presence of point mutation(s) closely associated with the fourth of these aligned regions. The murF2 allele, expressed and purified as a glutathione S-transferase::MurF2 fusion, was 181-fold less catalytically active at 30 degrees C and was further reduced at the nonpermissive temperature (42 degrees C). Thus the murF2 temperature-sensitive phenotype arises from a point mutation within a highly conserved region within this protein family. These data argue that these proteins comprise a superfamily of three substrate amide ligases that share significant structural and catalytic homologies.

The development of the structure-activity studies leading to the discovery of anacetrapib is described. These studies focused on varying the substitution of the oxazolidinone ring of the 5-aryloxazolidinone system. Specifically, it was found that substitution of the 4-position with a methyl group with the cis-stereochemistry relative to the 5-aryl group afforded compounds with increased cholesteryl ester transfer protein (CETP) inhibition potency and a robust in vivo effect on increasing HDL-C levels in transgenic mice expressing cynomolgus monkey CETP.