Conserved cytoplasmic motifs that distinguish sub-groups of the polyprenol phosphate:N-acetylhexosamine-1-phosphate transferase family.

WecA, MraY and WbcO are conserved members of the polyprenol phosphate:N-acetylhexosamine-1-phosphate transferase family involved in the assembly of bacterial cell walls, and catalyze reactions involving a membrane-associated polyprenol phosphate acceptor substrate and a cytoplasmically located UDP-D-amino sugar donor. MraY, WbcO and WecA purportedly utilize different UDP-sugars, although the molecular basis of this specificity is largely unknown. However, domain variations involved in specificity are predicted to occur on the cytoplasmic side of the membrane, adjacent to conserved domains involved in the mechanistic activity, and with access to the cytoplasmically located sugar nucleotides. Conserved C-terminal domains have been identified that satisfy these criteria. Topological analyses indicate that they form the highly basic, fifth cytoplasmic loop between transmembrane regions IX and X. Four diverse loops are apparent, for MraY, WecA, WbcO and RgpG, that uniquely characterize these sub-groups of the transferase family, and a correlation is evident with the known or implied UDP-sugar specificity.

One-day enzymatic synthesis and purification of UDP-N- [1-14C]acetyl-glucosamine.

UDP-GlcN[1-14C]Ac was synthesized in a single enzymatic reaction from [1-14C]acetate and commercially available precursors on both a microcurie (micromole) and a millicurie (millimole) scale. The reaction was catalyzed by the action of acetyl coenzyme A synthetase, inorganic pyrophosphatase, and the bifunctional Escherichia coli GlmU protein. Within 2 h 86 to 94% reaction is attained, and it approaches 99% completion overnight. GlmU protein was prepared in the form of a fusion suitable for nickel chelate affinity chromatography. Several methods were developed for rapid purification of UDP-GlcN[1-14C]Ac: an HPLC method handled micromole (microcurie) loads. Alternatively, ion exchange chromatography over DOWEX AG1 X-2 using a batch elution procedure was compatible with millimole (millicurie) amounts of radiolabel and yielded both chemically and radiochemically homogeneous UDP-GlcN[1-14C]Ac. These methods allow laboratories to quickly produce and purify microcurie to millicurie quantities of N-acetyl-labeled UDP-GlcNAc by a choice of methods from relatively inexpensive precursors.

Conditionally lethal Escherichia coli murein mutants contain point defects that map to regions conserved among murein and folyl poly-gamma-glutamate ligases: identification of a ligase superfamily.

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.

Cloning, expression, and purification of UDP-3-O-acyl-GlcNAc deacetylase from Pseudomonas aeruginosa: a metalloamidase of the lipid A biosynthesis pathway.

The lpxC (envA) gene of Escherichia coli encodes UDP-3-O-acyl-GlcNAc deacetylase, the second and committed step of lipopolysaccharide biosynthesis. Although present in all gram-negative bacteria examined, the deacetylase from E. coli is the only example of this enzyme that has been expressed and purified. In order to examine other variants of this protein, we cloned the Pseudomonas aeruginosa deacetylase structural gene from a lambda library as a 5.1-kb EcoRI fragment. The LpxC reading frame encodes an inferred protein of 33,435 Da that is highly homologous to the E. coli protein and that possesses a nearly identical hydropathy profile. In order to verify function, we subcloned the P. aeruginosa lpxC gene into the T7-based expression vector pET11a. Upon induction at 30 degrees C, this construct yielded active protein to approximately 18% of the soluble fraction. We devised a novel, rapid, and reproducible assay for the deacetylase which facilitated purification of the enzyme in three steps. The purified recombinant protein was found to be highly sensitive to EDTA yet was reactivated by the addition of excess heavy metal, as was the case for crude extracts of P. aeruginosa. In contrast, deacetylase activity in crude extracts of E. coli was insensitive to EDTA, and the extracts of the envA1 mutant were sensitive in a time-dependent manner. The lpxC gene has no significant homology with amidase signature sequences. Therefore, we assign this protein to the metalloamidase family as a member with a novel structure.

Kinetic mechanism of the Escherichia coli UDPMurNAc-tripeptide D-alanyl-D-alanine-adding enzyme: use of a glutathione S-transferase fusion.

The D-alanyl-D-alanine-adding enzyme encoded by the murF gene catalyzes the ATP-dependent formation of UDP-N-acetylmuramyl-L-gamma-D-Glu-meso-diaminopimelyl-D-Ala-D-Ala (UDP-MurNAc-tripeptide). MurF has been cloned from Escherichia coli and expressed as a glutathione S-transferase (GST) fusion using the tac promoter-based pGEX-KT vector. From induced, broken cell preparations, highly active fusion was recovered and purified in one step by affinity chromatography. The purified fusion protein was strongly inhibited by substrate UDPMurNAc-tripeptide, a response unaltered by changes in assay pH or by cleavage from the fusion partner. However, this effect was suppressed by the addition of 0.5 M NaCl. Initial velocity and dead-end inhibitor studies with the fusion enzyme were most consistent with a sequential ordered kinetic mechanism for the forward reaction in which ATP binds to free enzyme, followed by tripeptide and D-Ala-D-Ala in sequence prior to product release. Reported homologies between the MurF protein and the three preceding steps of cytoplasmic murein biosynthesis, MurC, -D, and -E, [Ikeda et al. (1990) J. Gen. Appl. Microbiol. 36, 179-187], raise the prospect that all of these enzymes will be found to proceed via this mechanism.

Regulation of UDP-3-O-[R-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase in Escherichia coli. The second enzymatic step of lipid a biosynthesis.

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 Km for the substrate, but is mainly an effect on Vmax. 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.

The envA permeability/cell division gene of Escherichia coli encodes the second enzyme of lipid A biosynthesis. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase.

The envA gene of Escherichia coli has been shown previously to be essential for cell viability (Beall, B. and Lutkenhaus, J. (1987) J. Bacteriol. 169, 5408-5415), yet it encodes a protein of unknown function. Extracts of strains harboring the mutant envA1 allele display 3.5-18-fold reductions in UDP-3-O-acyl-N-acetylglucosamine deacetylase specific activity. The deacetylase is the second enzymatic step of lipid A biosynthesis. The structural gene coding for the deacetylase has not been assigned. In order to determine if the envA gene encodes the deacetylase, envA was cloned into an isopropyl-1-thio-beta-D-galactopyranoside-inducible T7-based expression system. Upon induction, a protein of the size of envA was highly overproduced, as judged by SDS-PAGE. Direct deacetylase assays of cell lysates revealed a concomitant approximately 5,000-fold overproduction of activity. Assays of the purified, overproduced EnvA protein demonstrated a further approximately 5-fold increase in specific activity. N-terminal amino acid sequencing of the purified protein showed that the first 20 amino acids matched the predicted envA nucleotide sequence. Contaminating species were present at less than 1% of the level of the EnvA protein. Thus, envA is the structural gene for UDP-3-O-acyl-GlcNAc deacetylase. Based on its function in lipid A biosynthesis, we propose the new designation lpxC for this gene.

An Escherichia coli gene (FabZ) encoding (3R)-hydroxymyristoyl acyl carrier protein dehydrase. Relation to fabA and suppression of mutations in lipid A biosynthesis.

Escherichia coli strain SM101 harbors a temperature-sensitive allele (lpxA2) of the gene encoding UDP-Glc-NAc acyltransferase (the first enzyme of the lipid A pathway). SM101 is temperature-sensitive for lipid A biosynthesis and growth. To determine whether or not E. coli mutants lacking lipid A can be isolated, we examined temperature-resistant revertants of SM101. All regained the ability to synthesize lipid A. However, some were not true revertants but had acquired mutations in a neighboring gene (orf17), while retaining the original lpxA2 lesion. Cell extracts of such revertants displayed 2-5 fold reductions in the specific activity of (3R)-hydroxymyristoyl-ACP dehydrase. Wild-type cells that overproduced the protein encoded by orf17 overproduced (3R)-hydroxymyristoyl-ACP dehydrase activity as much as 170-fold, suggesting that orf17 is the structural gene for the dehydrase. The proposed function of orf17 is further supported by its sequence similarity to fabA, the structural gene for (3R)-hydroxydecanoyl dehydrase of E. coli. We suggest that bypass of the lpxA2 phenotype by mutations in orf17 may be due to an increased (3R)-hydroxymyristoyl-ACP pool. The orf17 gene (which we now designate fabZ) is not regulated by fadR. However, orf17 may be related to sefA, a suppressor of certain lesions in the cell division/lipid A biosynthesis gene, envA.