Conditionally Lethal Escherichia coli Murein Mutants Contain Point Defects That Map to Regions Conserved among Murein and Folyl Poly-γ-glutamate Ligases:  Identification of a Ligase Superfamily

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.

Biosynthesis of the cyanobacterial reserve polymer multi‐L‐arginyl‐poly‐L‐aspartic acid (cyanophycin)

“…As discussed previously [16], the C-terminal region (starting approximately at amino-acid residue 390; region overlined in Fig. 1) shows sequence similarity to members of a superfamily of peptide ligases [30,31] that includes certain murein ligases and folyl poly-gglutamate ligase. Members of this superfamily contain an ATP-binding P-loop motif, and show a Rossman fold [32].…”

The MurE Synthetase from Thermotoga maritima Is Endowed with an Unusual d-Lysine Adding Activity

“…The assembly of the peptide part of the monomer unit is ensured by a series of enzymes (MurC, MurD, MurE, and MurF) called the Mur synthetases. It has been shown that the Mur synthetases constitute a family of enzymes with common mechanistic and structural features (2,3).…”

atxAControlsBacillus anthracisCapsule Synthesis viaacpAand a Newly Discovered Regulator,acpB

“…The YwsC protein is similar to enzymes belonging to the ADP-forming amide bond ligase family. For enzymes of this type, ADP and P i are detected as end products of the polymerization reaction (13). The B. anthracis CapB protein is predicted to have four domains found in ADP-forming amide bond ligases and has significant amino acid homology to proteins of this class (13).…”

“…For enzymes of this type, ADP and P i are detected as end products of the polymerization reaction (13). The B. anthracis CapB protein is predicted to have four domains found in ADP-forming amide bond ligases and has significant amino acid homology to proteins of this class (13). The CapC homolog YwtA was found to be essential for polyglutamic acid polymer formation in B. subtilis natto since deletion of ywtA eliminates the ability of this strain to secrete the polymers.…”