Catalytic Mechanism of the Streptomyces K15 dd-Transpeptidase/Penicillin-Binding Protein Probed by Site-Directed Mutagenesis and Structural Analysis^,

Abstract

The Streptomyces K15 penicillin-binding DD-transpeptidase is presumed to be involved in peptide cross-linking during bacterial cell wall peptidoglycan assembly. To gain insight into the catalytic mechanism, the roles of residues Lys38, Ser96, and Cys98, belonging to the structural elements defining the active site cleft, have been investigated by site-directed mutagenesis, biochemical studies, and X-ray diffraction analysis. The Lys38His and Ser96Ala mutations almost completely abolished the penicillin binding and severely impaired the transpeptidase activities while the geometry of the active site was essentially the same as in the wild-type enzyme. It is proposed that Lys38 acts as the catalytic base that abstracts a proton from the active serine Ser35 during nucleophilic attack and that Ser96 is a key intermediate in the proton transfer from the Ogamma of Ser35 to the substrate leaving group nitrogen. The role of these two residues should be conserved among penicillin-binding proteins containing the Ser-Xaa-Asn/Cys sequence in motif 2. Conversion of Cys98 into Asn decreased the transpeptidase activity and increased hydrolysis of the thiolester substrate and the acylation rate with most beta-lactam antibiotics. Cys98 is proposed to play the same role as Asn in motif 2 of other penicilloyl serine transferases in properly positioning the substrate for the catalytic process.

A New Family of Cyanobacterial Penicillin-binding Proteins

“…Following passage through a 0.22-m filter, the protein mixture was applied to a Ni . Hydrolysis of 0.3 mM S2a, racemic S2c and S2d, and diastereoisomeric PhacATl thiolester substrates was performed at 37°C and monitored spectrophotometrically at 250 nm (22)(23)(24). Kinetic parameters were extracted by numerical simulation of the progression curves using the following equations,…”

“…A lower specificity observed with the racemic glycyl-thio-DL-lactate S2c substrate indicates a more tolerant recognition on the leaving group side. The hydrolysis efficiency of thiolester substrates is very variable among PBPs; the hydrolysis rates and k cat /K m were lower for Streptomyces K15 DD-peptidase, which is homologous to class A ␤-lactamases; and the catalytic activity of PBP-A was in the same range as Streptomyces R61, but the K m values were smaller (22,35). Compared with TEM-1 ␤-lactamase, PBP-A features similar activity on glycyl substrates (S2c and S2a) but is 350-fold more active on the S2d D-alanyl substrate, indicating a key difference in the specificity for the penultimate D-Ala.…”

Crystal Structures of the Apo and Penicillin-acylated Forms of the BlaR1 β-Lactam Sensor of Staphylococcus aureus

“…7A) (51). Deprotonation of the lysine could be accomplished through the relatively hydrophobic environment of the Lys 392 side chain consisting of Leu 395 , Met 434 , Phe 442 , Trp 475 , and Met 476 .…”

Design, synthesis, and structure–activity relationship study of 5-amido-1-(2,4-dinitrophenyl)-1H-4-pyrazolecarbonitrils as DD-carboxypeptidase/penicillin-binding protein inhibitors with Gram-positive antibacterial activity

“…The results of Table 2 show that these compounds can inhibit b-lactamase activity. Considering the SAR results and also the similarity between the active site of DD-peptidases and b-lactamases (Massova and Mobashery, 1998;Rhazi et al, 2003;Nicola et al, 2005;Kishida et al, 2006;Fisher et al, 2005), these results can provide good proof for the affinity of these compounds towards PBPs.…”

“…2). The similarity in the three-dimensional structure of the carboxypeptidase/ transpeptidase domains of PBPs is also matched by high degree of similarity in the reactive position of residues from three highly conserved motifs (Massova and Mobashery, 1998;Rhazi et al, 2003;Nicola et al, 2005;Kishida et al, 2006;Fisher et al, 2005). The first motif is the strictly conserved SXXK tetrad.…”