CLONING AND EXPRESSION OF STAPHYLOCOCCUS-AUREUS AND STREPTOCOCCUS-PYOGENES MURD GENES ENCODING URIDINE-DIPHOSPHATE N-ACETYLMURAMYL-L-ALANINE-D-GLUTAMATE LIGASES

Bacterial UDP-N-acetylmuramyl-L-alanine:D-glutamate ligase (MurD), a c ytoplasmic peptidoglycan biosynthetic enzyme, catalyzes the ATP-depend ent addition of D-glutamate to an alanyl residue of the UDP-N-acetylmu ramyl-L-alanine precursor: generating the dipeptide. The murD gene was cloned from both Staphylococcus aureus and Streptococcus pyogenes. Se quence analysis of the S. aureus murD gene revealed an open reading fr ame of 449 amino acids. The deduced aa sequence of S. aureus MurD is h ighly homologous to MurD from Escherichia coli, Haemophilus influencae , Bacillus subtilis and St. pyogenes. Recombinant MurD protein from bo th S. aureus and St. pyogenes was separately overproduced in E. coli a nd purified as His-tagged fusion. Both recombinant enzymes catalyzed t he ATP-dependent addition of D-glutamate to the precursor sugar peptid e. (C) 1998 Elsevier Science B.V.