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 diphos pho-N-acetylmuramic acid (UDP-MurNAc). By comparing the amino acid seq uences of MurC, -D, -E, and -F proteins from various bacterial genera, four regions of homology were identified. A profile search of Swisspr ot for related sequences revealed that these regional similarities wer e present in the folyl-gamma-polyglutamate ligases. These sequence hom ologies appear to track with catalytic function: both enzyme families proceed through an ordered kinetic mechanism and form product via an a cyl phosphate intermediate. Two highly conserved residues in region II were examined through site-directed mutagenesis of the murein D-alany l-D-alanine-adding enzyme from Escherichia coli (murF; E158 and H188). All mutations were highly detrimental to activity with enzyme specifi c activity reductions of 200-4500-fold, validating the critical nature of these residues. DNA sequence analysis from three E. coli mutants h arboring the murC3 (G344D), murE1 (G344K, A495S), and murF2 (A288T) mu tations 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-fol d less catalytically active at 30 degrees C and was further reduced at the nonpermissive temperature (42 degrees C). Thus the murF2 temperat ure-sensitive phenotype arises from a point mutation within a highly c onserved region within this protein family. These data argue that thes e proteins comprise a superfamily of three substrate amide ligases tha t share significant structural and catalytic homologies.