Gene 210 (1998) 117–125 Cloning and expression of Staphylococcus aureus and Streptococcus pyogenes murD genes encoding uridine diphosphate N-acetylmuramoyl--alanine:-glutamate ligases Mohamed El-Sherbeini *, Wayne M. Geissler, Jamya Pittman, Xiling Yuan, Kenny K. Wong, David L. Pompliano Department of Enzymology, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA Received 17 September 1997; received in revised form 7 January 1998; accepted 7 January 1998; Received by J. Wild Abstract Bacterial UDP-N-acetylmuramyl--alanine:-glutamate ligase (MurD), a cytoplasmic peptidoglycan biosynthetic enzyme, catalyzes the ATP-dependent addition of -glutamate to an alanyl residue of the UDP-N-acetylmuramyl--alanine precursor, generating the dipeptide. The murD gene was cloned from both Staphylococcus aureus and Streptococcus pyogenes. Sequence analysis of the S. aureus murD gene revealed an open reading frame of 449 amino acids. The deduced aa sequence of S. aureus MurD is highly homologous to MurD from Escherichia coli, Haemophilus influenzae, Bacillus subtilis and St. pyogenes. Recombinant MurD protein from both S. aureus and St. pyogenes was separately overproduced in E. coli and purified as His-tagged fusion. Both recombinant enzymes catalyzed the ATP-dependent addition of -glutamate to the precursor sugar peptide. © 1998 Elsevier Science B.V. Keywords: Bacterial cell wall; Peptidoglycan; Gram-positive cocci 1. Introduction dependent amino acid ligases (MurC, -D, -E, and -F ) catalyze the stepwise synthesis of the pentapeptide sidechain using the newly synthesized lactyl carboxylate The bacterial cell wall is a polymer —a single molecule as the first acceptor site. Attachment of the sugar composed of peptidoglycan—that defines the boundary pentapeptide to a lipid carrier in the plasma membrane and shape of the cell. Assembled by cross-linking glycan and addition of another glucosamine unit to the 4-OH chains with short peptide bridges (Rogers et al., 1980), of the muramic acid moiety completes the synthesis of the completed structure is strong enough to maintain the monomeric building block. The finished precursor cell integrity against an osmotic pressure differential of is moved across the membrane into the periplasm where over four atmospheres, but also flexible enough to allow it is stitched enzymatically by the penicillin-binding the cell to move, grow and divide. The construction of proteins into the fabric of the growing cell wall. the peptidoglycan begins in the cytoplasm with an Because the pentapeptide sidechain is not synthesized activated sugar molecule, UDP-N-acetylglucosamine. ribosomally, it contains a more diverse chemical func- After two reactions (catalyzed by MurA and MurB) tionality than a typical peptide, both structurally and that result in the placement of a lactyl group on the stereochemically. Two of the enzymes catalyze the addi- 3-OH of the glucosamine moiety, a series of ATP- tion of -amino acids (MurD and MurF ), and MurE mediates the formation of a peptide bond between the c-carboxylate of -glutamate and the amino group of * Corresponding author. Tel: +1 908 594 5586; Fax: +1 908 594 5878; e-mail: Mohamed_El-Sherbeini@merck.com -lysine. Presumably, these structures render the exposed peptidoglycan resistant to the action of proteases, but Abbreviations: aa, amino acid(s); IPTG, isopropyl b--thiogalactopyr- they also imply that the active sites of the enzymes must anoside; kb, kilobases; nt, nucleotide(s); PAGE, polyacrylamide-gel have unusual structures in order to handle the somewhat electrophoresis; PBS, phosphate-buffered saline; PCR, polymerase uncommon substrates. It is into these unusual active chain reaction; S., Staphylococcus; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCl/0.015 M Na 3 .citrate pH 7.6; St., Streptococcus; u, unit(s). sites that we hope to bind novel inhibitors. 0378-1119/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0378-1119(98)00059-6