Staphylococcus aureus sortase (SrtA) is a thiol transpeptidase. The enzyme catalyzes a cell wall sorting reaction in which a surface protein with a sorting signal containing a LPXTG motif is cleaved between the threonine and glycine residues. The resulting threonine carboxyl end of this protein is covalently attached to a pentaglycine cross-bridge of peptidoglycan. The transpeptidase activity of sortase has been demonstrated in in vitro reactions between a LPETG-containing peptide and triglycine. When a nucleophile is not available, sortase slowly hydrolyzes the LPETG peptide at the same site. In this study, we have analyzed the steady-state kinetics of these two types of reactions catalyzed by sortase. The kinetic results fully support a ping-pong mechanism in which a common acyl-enzyme intermediate is formed in transpeptidation and hydrolysis. However, each reaction has a distinct rate-limiting step: the formation of the acyl-enzyme in transpeptidation and the hydrolysis of the same acyl-enzyme in the hydrolysis reaction. We have also demonstrated in this study that the nucleophile binding site of S. aureus sortase SrtA is specific for diglycine. While S1' and S2' sites of the enzyme both prefer a glycine residue, the S1' site is exclusively selective for glycine. Lengthening of the polyglycine acceptor nucleophile beyond diglycine does not further enhance the binding and catalysis.

An efficient "one-step" synthesis of cyclic amidines and guanidines has been developed. Treatment of cyclic amides and ureas with benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), base, and nitrogen nucleophiles leads to the formation of the corresponding cyclic amidines and guanidines, typically in good to excellent yields. This method has also been used to prepare heteroaryl ethers and thioethers using phenol and thiophenol nucleophiles. Time course NMR and HPLC-MS studies have facilitated explicit characterization of the proposed intermediates (the phosphonium salt and HOBt adduct); the data reveal a stepwise reaction pathway.

The enzymatic synthesis of the complete L-alanyl 1 -Lalanine 2 side chain of the peptidoglycan precursors of Enterococcus faecalis was obtained in vitro using purified enzymes. The pathway involved alanyl-tRNA synthetase and two ligases, BppA1 and BppA2, that specifically transfer alanine from Ala-tRNA to the first and second positions of the side chain, respectively. The structure of the UDP-N-acetylmuramoyl-L-Ala-␥-D-Glu-LLys(N ⑀ -L-Ala 1 -L-Ala 2 )-D-Ala-D-Ala product of BppA1 and BppA2 was confirmed by mass spectrometry (MS) and MS/MS analyses. The peptidoglycan structure of the wild-type E. faecalis strain JH2-2 was determined by tandem reverse-phase high-pressure liquid chromatography-MS revealing that most muropeptides contained two L-alanyl residues in the cross-bridges and in the free N-terminal ends. Deletion of the bppA2 gene was associated with production of muropeptides containing a single alanyl residue at these positions. The relative abundance of monomers, dimers, trimers, and tetramers in the peptidoglycan of the bppA2 mutant indicated that precursors containing an incomplete side chain were efficiently used by the DD-transpeptidases in the crosslinking reaction. However, the bppA2 deletion impaired expression of intrinsic ␤-lactam resistance suggesting that the low affinity penicillin-binding protein 5 did not function optimally with precursors substituted by a single alanine.Variations in the structure of peptidoglycan from Grampositive bacteria involve mainly the third and fifth (C-terminal) positions of the pentapeptide stem linked to the disaccharide GlcNAc-MurNAc 1 (see Fig. 1 (Fig. 1). Factors essential for methicillin resistance (fem) in S. aureus include the femA and femB genes that are required for synthesis of the pentaglycine side chain of the peptidoglycan precursors (4).In the late 1960s, the ligases for addition of glycine and L-amino acids to the ⑀-amino group of L-lysine were shown to use aminoacyl-tRNAs as substrates, whereas D-amino acids are added in a tRNA-independent reaction (5, 6). More recently, the ligase for incorporation of the first L-alanine of the side chain of the E. faecalis (7) and Weissella viridescens (8) peptidoglycan precursors has been identified based on cloning of fem-related genes in Escherichia coli and assays of the purified gene products for UDP-MurNAc-pentapeptide:L-alanine ligase activity. The assay developed in our laboratory relies on in vitro synthesis of Ala-tRNA by the purified E. faecalis alanyl-tRNA synthetase and subsequent transfer of L-Ala from the aminoacyl-tRNA to the nucleotide precursor UDP-MurNAc-L-Ala-␥-D-Glu-L-Lys-D-Ala-D-Ala (UDP-MurNAc-pentapeptide) (7). In the current study, we used this approach to identify the ligase for incorporation of the second L-alanine in the side chain of E. faecalis peptidoglycan precursors. We report in vitro synthesis of the complete L-alanyl-L-alanine side chain, deletion of the gene encoding the ligase for incorporation of the second L-alanine from the chromosome of E. faecalis JH2-2, and the analys...

Recently, for the first time in the history of this bacterial species, methicillin-resistant Staphylococcus aureus (MRSA) carrying the enterococcal vanA gene complex and expressing high level resistance to vancomycin was identified in clinical specimens (CDC (2002) MMWR 51, 565-567). The purpose of our studies was to understand how vanA is expressed in the heterologous background of S. aureus and how it interacts with the mecAbased resistance mechanism, which is also present in these strains and is targeted on cell wall biosynthesis. The vanA-containing staphylococcal plasmid was transferred from the clinical vancomycin-resistant S. aureus (VRSA) strain HIP11714 (CDC (2002) MMWR 51, 565-567) to the methicillin-resistant S. aureus (MRSA) strain COL for which extensive genetic and biochemical information is available on staphylococcal cell wall biochemistry and drug resistance mechanisms. The transconjugant named COLVA showed high and homogeneous resistance to both oxacillin and vancomycin. COLVA grown in vancomycin-containing medium produced an abnormal peptidoglycan: all pentapeptides were replaced by tetrapeptides, and the peptidoglycan contained at least 22 novel muropeptide species that frequently showed a deficit or complete absence of pentaglycine branches. The UDP-MurNAc-pentapeptide, the major component of the cell wall precursor pool in vancomycin-sensitive cells was replaced by UDPMurNAc-depsipeptide and UDP-MurNAc-tetrapeptide. Transposon inactivation of the ␤-lactam resistance gene mecA caused complete loss of ␤-lactam resistance but had no effect on the expression of vancomycin resistance. The two major antibiotic resistance mechanisms encoded by mecA and vanA residing in the same S. aureus appear to use different sets of enzymes for the assembly of cell walls.Until the late 1990s, clinical isolates of Saccharomyces aureus have retained a uniform high sensitivity to vancomycin with minimal inhibitory concentration (MIC) 1 values in the vicinity of 1 g/ml. Beginning with the late 1990s, isolates with reduced susceptibility to vancomycin began to be reported from several countries (1), but the MIC value of these vancomycin intermediate-resistant S. aureus isolates was limited to the range of 8 -16 g/ml of the antibiotic. The first highly vancomycin-resistant S. aureus (VRSA) isolates (MIC over 32 g/ml) were only detected during the last year in two hospitals in the United States (2, 3). The appearance of such VRSA strains in clinical specimens is of obvious and grave concern, because the spread of VRSA isolates may seriously jeopardize the chemotherapy of multidrug-resistant S. aureus disease and raise the specter of untreatable staphylococcal infections (4, 5). The VRSA strain HIP11714 recovered from a dialysis patient in Detroit, Michigan has acquired the vanA gene complex most likely from a vancomycin-resistant Enterococcus faecalis strain coinfecting the diabetic wound and catheter insertion site of the patient (1). HIP11714 was also methicillin-resistant: it carried the heterologous mecA gene, t...

Previous studies suggested that a Gly-containing branch of cell wall precursor [C 55 -MurNAc-(peptide)-GlcNAc], which is often referred to as lipid II, might serve as a nucleophilic acceptor in sortase-catalyzed anchoring of surface proteins in Staphylococcus aureus. To test this hypothesis, we first simplified the procedure for in vitro biosynthesis of Gly-containing lipid II by using branched UDP-MurNAc-hexapeptide isolated from the cytoplasm of Streptomyces spp. Second, we designed a thin-layer chromatography-based assay in which the mobility of branched but not linear lipid II is shifted in the presence of both sortase and LPSTG-containing peptide. These results and those of additional experiments presented in this study further suggest that lipid II indeed serves as a natural substrate in a sorting reaction.