Nucleotide sequences of the pbpX genes encoding the penicillin-binding proteins 2x from Streptococcus pneumoniae R6 and a cefotaxime-resistant mutant, C506

Development of penicillin resistance in Streptococcus pneumoniae is due to successive mutations in penicillin-binding proteins (PBPs) which reduce their affinity for beta-lactam antibiotics. PBP2x is one of the high-Mr PBPs which appears to be altered both in resistant clinical isolates, and in cefotaxime-resistant laboratory mutants. In this study, we have sequenced a 2564 base-pair chromosomal fragment from the penicillin-sensitive S. pneumoniae strain R6, which contains the PBP2x gene. Within this fragment, a 2250 base-pair open reading frame was found which coded for a protein having an Mr of 82.35kD, a value which is in good agreement with the Mr of 80-85 kD measured by SDS-gel electrophoresis of the PBP2x protein itself. The N-terminal region resembled an unprocessed signal peptide and was followed by a hydrophobic sequence that may be responsible for membrane attachment of PBP2x. The corresponding nucleotide sequence of the PBP2x gene from C504, a cefotaxime-resistant laboratory mutant obtained after five selection steps, contained three nucleotide substitutions, causing three amino acid alterations within the beta-lactam binding domain of the PBP2x protein. Alterations affecting similar regions of Escherichia coli PBP3 and Neisseria gonorrhoeae PBP2 from beta-lactam-resistant strains are known. The penicillin-binding domain of PBP2x shows highest homology with these two PBPs and S. pneumoniae PBP2b. In contrast, the N-terminal extension of PBP2x has the highest homology with E. coli PBP2 and methicillin-resistant Staphylococcus aureus PBP2'. No significant homology was detected with PBP1a or PBP1b of Escherichia coli, or with the low-Mr PBPs.     

Horizontal spread of an altered penicillin-binding protein 2B gene between Streptococcus pneumoniae and Streptococcus oralis

Abstract The region encoding the transpeptidase domain of the penicillin-binding protein 2B (PBP 2B) gene of two penicillin-resistant clinical isolates of Streptococcus oralis was > 99.6% identical in nucleotide sequence to that of a penicillin-resistant serotype 6 isolate of Streptococcus pneumoniae . The downstream 849 base pairs of these genes were identical. Analysis of the data indicates that the PBP gene has probably been transferred from S. pneumoniae into S. oralis , rather than vice versa, and shows that one region of this resistance gene has been distributed horizontally both within S. pneumoniae and into two different viridans group streptococci.     

Mutational analysis of the Streptococcus pneumoniae bimodular class A penicillin-binding proteins.

One group of penicillin target enzymes, the class A high-molecular-weight penicillin-binding proteins (PBPs), are bimodular enzymes. In addition to a central penicillin-binding-transpeptidase domain, they contain an N-terminal putative glycosyltransferase domain. Mutations in the genes for each of the three Streptococcus pneumoniae class A PBPs, PBP1a, PBP1b, and PBP2a, were isolated by insertion duplication mutagenesis within the glycosyltransferase domain, documenting that their function is not essential for cellular growth in the laboratory. PBP1b PBP2a and PBP1a PBP1b double mutants could also be isolated, and both showed defects in positioning of the septum. Attempts to obtain a PBP2a PBP1a double mutant failed. All mutants with a disrupted pbp2a gene showed higher sensitivity to moenomycin, an antibiotic known to inhibit PBP-associated glycosyltransferase activity, indicating that PBP2a is the primary target for glycosyltransferase inhibitors in S. pneumoniae.     

Growth and division of Streptococcus pneumoniae: localization of the high molecular weight penicillin-binding proteins during the cell cycle

The bacterial peptidoglycan, the main component of the cell wall, is synthesized by the penicillin-binding proteins (PBPs). We used immunofluorescence microscopy to determine the cellular localization of all the high molecular weight PBPs of the human pathogen Streptococcus pneumoniae, for a wild type and for several PBP-deficient strains. Progression through the cell cycle was investigated by the simultaneous labelling of DNA and the FtsZ protein. Our main findings are: (i) the temporal dissociation of cell wall synthesis, inferred by the localization of PBP2x and PBP1a, from the constriction of the FtsZ-ring; (ii) the localization of PBP2b and PBP2a at duplicated equatorial sites indicating the existence of peripheral peptidoglycan synthesis, which implies a similarity between the mechanism of cell division in bacilli and streptococci; (iii) the abnormal localization of some class A PBPs in PBP-defective mutants which may explain the apparent redundancy of these proteins in S. pneumoniae.     

Relatedness between Streptococcus pneumoniae and viridans streptococci: transfer of penicillin resistance determinants and immunological similarities of penicillin-binding proteins

The occurrence of highly variable penicillin-binding proteins (PBPs) in penicillin-resistant Streptococcus pneumoniae suggested that transfer of homologous genes from related species may be involved in resistance development. Antiserum and monoclonal antibodies raised against PBPs 1a and 2b from the susceptible S. pneumoniae R6 strain were used to identify related PBPs in 41 S. mitis, S. sanguis I and S. sanguis II strains mostly isolated in South Africa with MIC values ranging from less than 0.15 to 16 mg/ml. Furthermore, the possibility of genetic exchange was examined with 30 penicillin-resistant strains of this collection (MIC greater than 0.06 mg/ml) as donors using S. pneumoniae R6 as recipient in transformation experiments. The majority of S. mitis and S. sanguis II strains but none of the S. sanguis I strains could transform penicillin resistance genes into S. pneumoniae R6. All positive donor strains and all susceptible isolates of S. mitis and S. sanguis II strains contained PBPs which cross-reacted with the anti-PBP 1a and/or anti-PBP 2b antibodies. On the other hand, only five of the 14 S. sanguis I strains contained a PBP that reacted with one of the antibodies. This strongly suggested the presence of genes homologous to the pneumococcal PBP 1a and 2b genes in viridans streptococci, and documents that penicillin resistance determinants can be transformed from viridans streptococci into the pneumococcus.     

Molecular characteristics of penicillin-binding protein 2b, 2x, and 1a sequences in penicillin-nonsusceptible Streptococcus pneumoniae isolates in Shenyang, China

The aim of this study was to investigate the nature of the amino acid motifs found in penicillin-binding proteins (PBP) 2b, 2x, and 1a of penicillin-nonsusceptible Streptococcus pneumoniae isolates from Shenyang, China, and to obtain information regarding the prevalence of alterations within the motifs or in positions flanking the motifs. For 18 clinical isolates comprising 4 penicillin-susceptible S. pneumoniae, 5 penicillin-intermediate S. pneumoniae, and 9 penicillin-resistant S. pneumoniae. the DNA sequences of PBP2b, PBP2x, and PBP1a transpeptidase domains were determined and then genotyped by multilocus sequence typing. Sequence analysis revealed that most penicillin-nonsusceptible S. pneumoniae isolates (penicillin MIC > or = 1.5 microg/mL and cefotaxime MIC > or = 2 microg/mL) shared identical PBP2b, PBP2x, and PBP1a amino acid profiles. Most penicillin-resistant S. pneumoniae isolates were ST320 (4-16-19-15-6-20-1), the double-locus variant of the Taiwan19F-14 clone. This study will serve as a basis for future monitoring of genetic changes associated with the emergence and spread of beta-lactam resistance in Shenyang, China.     

Extensive re-modelling of the transpeptidase domain of penicillin-binding protein 2B of a penicillin-resistant South African isolate of Streptococcus pneumoniae

Clinical isolates of Streptococcus pneumoniae that have greatly increased levels of resistance to penicillin (greater than 1000-fold) have been reported from South Africa during the last ten years. Penicillin resistance in these strains is entirely due to the development of penicillin-binding proteins (PBPs) with decreased affinity for penicillin. We have cloned and sequenced the coding region for the transpeptidase domain of penicillin-binding protein 2B from three penicillin-sensitive strains of S. pneumoniae and from a penicillin-resistant South African strain. The amino acid sequences of the transpeptidase domains of PBP2B of the three penicillin-sensitive strains were identical and there were only between one and four differences in the nucleotide sequences of their coding regions. The corresponding region of the PBP2B gene from the penicillin-resistant strain differed by 74 nucleotide substitutions which resulted in 17 alterations in the amino acid sequence of PBP2B. The most remarkable alteration that has occurred during the development of the 'penicillin-resistant' form of PBP2B is the substitution of seven consecutive residues in a region that is predicted to form a loop at the bottom of the penicillin-binding site.     

Nucleotide sequence of the pbpA gene and characteristics of the deduced amino acid sequence of penicillin-binding protein 2 of Escherichia coli K12

We have determined the nucleotide sequence of the pbpA gene encoding penicillin-binding protein (PBP) 2 of Escherichia coli. The coding region for PBP 2 was 1899 base pairs in length and was preceded by a possible promoter sequence and two open reading frames. The primary structure of PBP 2, deduced from the nucleotide sequence, comprised 633 amino acid residues. The relative molecular mass was calculated to be 70867. The deduced sequence agreed with the NH2-terminal sequence of PBP 2 purified from membranes, suggesting that PBP 2 has no signal peptide. The hydropathy profile suggested that the NH2-terminal hydrophobic region (a stretch of 25 non-ionic amino acids) may anchor PBP 2 in the cytoplasmic membrane as an ectoprotein. There were nine homologous segments in the amino acid sequence of PBP 2 when compared with PBP 3 of E. coli. The active-site serine residue of PBP 2 was predicted to be Ser-330. Around this putative active-site serine residue was found the conserved sequence of Ser-Xaa-Xaa-Lys, which has been identified in all of the other E. coli PBPs so far studied (PBPs 1A, 1B, 3, 5 and 6) and class A and class C beta-lactamases. In the higher-molecular-mass PBPs 1A, 1B, 2 and 3, Ser-Xaa-Xaa-Lys-Pro was conserved. In the putative peptidoglycan transpeptidase domain there were six amino acid residues, which are common only in the PBPs of higher molecular mass.     

Crystal structure of penicillin-binding protein 1a (PBP1a) reveals a mutational hotspot implicated in beta-lactam resistance in Streptococcus pneumoniae

Streptococcus pneumoniae is a major human pathogen whose infections have been treated with beta-lactam antibiotics for over 60 years, but the proliferation of strains that are highly resistant to such drugs is a problem of worldwide concern. Beta-lactams target penicillin-binding proteins (PBPs), membrane-associated enzymes that play essential roles in the peptidoglycan biosynthetic process. Bifunctional PBPs catalyze both the polymerization of glycan chains (glycosyltransfer) and the cross-linking of adjacent pentapeptides (transpeptidation), while monofunctional enzymes catalyze only the latter reaction. Although S. pneumoniae has six PBPs, only three (PBP1a, PBP2x, PBP2b) are major resistance determinants, with PBP1a being the only bifunctional enzyme. PBP1a plays a key role in septum formation during the cell division cycle and its modification is essential for the development of high-level resistance to penicillins and cephalosporins. The crystal structure of a soluble form of pneumococcal PBP1a (PBP1a*) has been solved to 2.6A and reveals that it folds into three domains. The N terminus contains a peptide from the glycosyltransfer domain bound to an interdomain linker region, followed by a central, transpeptidase domain, and a small C-terminal unit. An analysis of PBP1a sequences from drug-resistant clinical strains in light of the structure reveals the existence of a mutational hotspot at the entrance of the catalytic cleft that leads to the modification of the polarity and accessibility of the mutated PBP1a active site. The presence of this hotspot in all variants sequenced to date is of key relevance for the development of novel antibiotherapies for the treatment of beta-lactam-resistant pneumococcal strains.     

Glycosyltransferase domain of penicillin-binding protein 2a from Streptococcus pneumoniae is membrane associated

Penicillin-binding proteins (PBPs) are bacterial cytoplasmic membrane proteins that catalyze the final steps of the peptidoglycan synthesis. Resistance to beta-lactams in Streptococcus pneumoniae is caused by low-affinity PBPs. S. pneumoniae PBP 2a belongs to the class A high-molecular-mass PBPs having both glycosyltransferase (GT) and transpeptide (TP) activities. Structural and functional studies of both domains are required to unravel the mechanisms of resistance, a prerequisite for the development of novel antibiotics. The extracellular region of S. pneumoniae PBP 2a has been expressed (PBP 2a*) in Escherichia coli as a glutathione S-transferase fusion protein. The acylation kinetic parameters of PBP 2a* for beta-lactams were determined by stopped-flow fluorometry. The acylation efficiency toward benzylpenicillin was much lower than that toward cefotaxime, a result suggesting that PBP 2a participates in resistance to cefotaxime and other beta-lactams, but not in resistance to benzylpenicillin. The TP domain was purified following limited proteolysis. PBP 2a* required detergents for solubility and interacted with lipid vesicles, while the TP domain was water soluble. We propose that PBP 2a* interacts with the cytoplasmic membrane in a region distinct from its transmembrane anchor region, which is located between Lys 78 and Ser 156 of the GT domain.     

Streptococcus faecium mutants that are temperature sensitive for cell growth and show alterations in penicillin-binding proteins.

The penicillin-binding proteins (PBPs) of 209 cell division (or growth) temperature-sensitive mutants of Streptococcus faecium were analyzed in this study. A total of nine strains showed either constitutive or temperature-sensitive conditional damage in the PBPs. Analysis of these nine strains yielded the following results: one carried a PBP 1 constitutively showing a lower molecular weight; one constitutively lacked PBP 2; two lacked PBP 3 at 42 degrees C, but not at 30 degrees C; one was normal at 30 degrees C but at 42 degrees C lacked PBP 3 and overproduced PBP 5; two were normal at 42 degrees C and lacked PBP 5 at 30 degrees C; one constitutively lacked PBP 5; and one carried a PBP 6 constitutively split in two bands. The mutant lacking PBP 3 and overproducing PBP 5 continued to grow at 42 degrees C for 150 min and then lysed. Revertants selected for growth capability at 42 degrees C from the mutants altered in PBPs 5 and 6 maintained the same PBP alterations, while those isolated from the strains with altered PBP 1 or lacking PBP 2 or PBP 3 showed a normal PBP pattern. Penicillin-resistant derivatives were isolated at 30 degrees C from the mutants lacking PBP 2 and from that lacking PBP 3. All these derivatives continued to show the same PBP damage as the parents, but overproduced PBP 5 and grew at 42 degrees C. These findings indicate that high-molecular-weight, but not low-molecular-weight, PBPs are essential for cell growth in S. faecium. This is in complete agreement with previous findings obtained with a different experimental system. On the basis of both previous and present data it is suggested that PBPs 1, 2, and 3 appear necessary for cell growth at optimal temperature (and at maximal rate), but not for cell growth at a submaximal one (or at a reduced rate), and an overproduced PBP 5 is capable of taking over the function of PBPs 1, 2, and 3.     

Unusual septum formation in Streptococcus pneumoniae mutants with an alteration in the D,D-carboxypeptidase penicillin-binding protein 3.

An internal 630-bp DNA fragment of the gene encoding penicillin-binding protein 3 (PBP 3) (dacA) of Streptococcus pneumoniae was identified in a lambda gt11 gene bank screened with anti-PBP 3 antiserum. The deduced 210-amino-acid sequence showed a high degree of homology to the low-molecular-weight PBPs 5 and 6 of Escherichia coli and Bacillus subtilis PBP 5. Viable mutants lacking a C-terminal part of PBP 3 were obtained after a plasmid containing the dacA fragment was integrated into the PBP 3 gene by homologous recombination. The truncated PBP 3* was still active in terms of beta-lactam binding. Most PBP 3 was found in the growth medium, indicating that membrane anchoring of PBP 3 is provided by the C terminus, as has been shown for other D,D-carboxypeptidases. The mutant cells grew with a slower generation time than the wild type in the shape of irregular enlarged spheres. In addition, as revealed by electron microscopy, cell separation was severely affected, septa were found unevenly distributed at multiple sites within the cells, and the murein layer appeared variable in thickness.     

Structural and mechanistic basis of penicillin-binding protein inhibition by lactivicins

Beta-lactam antibiotics, including penicillins and cephalosporins, inhibit penicillin-binding proteins (PBPs), which are essential for bacterial cell wall biogenesis. Pathogenic bacteria have evolved efficient antibiotic resistance mechanisms that, in Gram-positive bacteria, include mutations to PBPs that enable them to avoid beta-lactam inhibition. Lactivicin (LTV; 1) contains separate cycloserine and gamma-lactone rings and is the only known natural PBP inhibitor that does not contain a beta-lactam. Here we show that LTV and a more potent analog, phenoxyacetyl-LTV (PLTV; 2), are active against clinically isolated, penicillin-resistant Streptococcus pneumoniae strains. Crystallographic analyses of S. pneumoniae PBP1b reveal that LTV and PLTV inhibition involves opening of both monocyclic cycloserine and gamma-lactone rings. In PBP1b complexes, the ring-derived atoms from LTV and PLTV show a notable structural convergence with those derived from a complexed cephalosporin (cefotaxime; 3). The structures imply that derivatives of LTV will be useful in the search for new antibiotics with activity against beta-lactam-resistant bacteria.     

Common antigenic determinants of pneumococcal penicillin-binding protein (PBP) 1a and Streptococcus pyogenes PBP 2

Abstract Antisera raised against penicillin-binding protein (PBP) 1a of Streptococcus pneumoniae reacted with PBP 2 in certain strains of Streptococcus pyogenes . Cross-reactivity could be demonstrated on immunoblots as well as by immunoprecipitation of native solubilised proteins, indicating a similar structural arrangement also in the native form of the two PBPs.     

Five independent combinations of mutations can result in low-affinity penicillin-binding protein 2x of Streptococcus pneumoniae.

Penicillin-binding protein 2x (PBP 2x) of Streptococcus pneumoniae is one of the high-molecular-weight PBPs involved in the development of intrinsic beta-lactam resistance. Point mutations in the PBP 2x genes (pbpX) have now been characterized in five independent spontaneous laboratory mutants in order to identify protein regions which are important for interaction with beta-lactam antibiotics. All mutant genes contained two to four mutations resulting in amino acid substitutions within the penicillin-binding domain of PBP 2x, and none of the mutants carried an identical set of mutations. For one particular mutant, C606, carrying four mutations in pbpX, the mutations at positions 601 and 597 conferred first- and second-level resistance when introduced into the susceptible parent strain S. pneumoniae R6. However, the other two mutations, at amino acid positions 289 and 422, which were originally selected at the fifth and sixth isolation steps, did not contribute at all to resistance in similar experiments. This suggests that they are phenotypically expressed only in combination with mutations in other genes. Three PBP 2x regions were mutated in from two to all four mutants carrying a low-affinity PBP 2x. However, in a fifth mutant containing a PBP 2x with apparent zero affinity for beta-lactams, the three mutations in pbpX mapped at entirely different positions. This demonstrates that different mutational pathways exist for remodeling this PBP during resistance development.     

Potential transition state analogue inhibitors for the penicillin-binding proteins

Penicillin-binding proteins (PBPs) are ubiquitous bacterial enzymes involved in cell wall biosynthesis. The development of new PBP inhibitors is a potentially viable strategy for developing new antibacterial agents. Several potential transition state analogue inhibitors for the PBPs were synthesized, including peptide chloromethyl ketones, trifluoromethyl ketones, aldehydes, and boronic acids. These agents were characterized chemically, stereochemically, and as inhibitors of a set of low molecular mass PBPs: Escherichia coli (EC) PBP 5, Neisseria gonorrhoeae (NG) PBP 3, and NG PBP 4. A peptide boronic acid was the most effective PBP inhibitor in the series, with a preference observed for a d-boroAla-based over an l-boroAla-based inhibitor, as expected given that physiological PBP substrates are based on d-Ala at the cleavage site. The lowest K(I) of 370 nM was obtained for NG PBP 3 inhibition by Boc-l-Lys(Cbz)-d-boroAla (10b). Competitive inhibition was observed for this enzyme-inhibitor pair, as expected for an active site-directed inhibitor. For the three PBPs included in this study, an inverse correlation was observed between the values for log K(I) with 10b and the values for log(k(cat)/K(m)) for activity against the analogous substrate, and K(m)/K(I) ratios were 90, 1900, and 9600 for NG PBP 4, EC PBP 5, and NG PBP 3, respectively. These results demonstrate that peptide boronic acids can be effective transition state analogue inhibitors for the PBPs and provide a basis for the use of these agents as probes of PBP structure, function, and mechanism, as well as a possible basis for the development of new PBP-targeted antibacterial agents.     

Functional characterization of penicillin-binding protein 1b from Streptococcus pneumoniae

The widespread use of antibiotics has encouraged the development of drug resistance in pathogenic bacteria. In order to overcome this problem, the modification of existing antibiotics and/or the identification of targets for the design of new antibiotics is currently being undertaken. Bifunctional penicillin-binding proteins (PBPs) are membrane-associated molecules whose transpeptidase (TP) activity is irreversibly inhibited by beta-lactam antibiotics and whose glycosyltransferase (GT) activity represents a potential target in the antibacterial fight. In this work, we describe the expression and the biochemical characterization of the soluble extracellular region of Streptococcus pneumoniae PBP1b (PBP1b*). The acylation efficiency for benzylpenicillin and cefotaxime was characterized by stopped-flow fluorometry and a 40-kDa stable TP domain was generated after limited proteolysis. In order to analyze the GT activity of PBP1b*, we developed an electrophoretic assay which monitors the fluorescence signal from PBP1b*-bound dansylated lipid II. This binding was inhibited by the antibiotic moenomycin and was specific for the GT domain, since no signal was observed in the presence of the purified functional TP domain. Binding studies performed with truncated forms of PBP1b* demonstrated that the first conserved motif of the GT domain is not required for the recognition of lipid II, whereas the second motif is necessary for such interaction.     

The glycosyltransferase domain of penicillin-binding protein 2a from Streptococcus pneumoniae catalyzes the polymerization of murein glycan chains

The bacterial peptidoglycan consists of glycan chains of repeating beta-1,4-linked N-acetylglucosaminyl-N-acetylmuramyl units cross-linked through short peptide chains. The polymerization of the glycans, or glycosyltransfer (GT), and transpeptidation (TP) are catalyzed by bifunctional penicillin-binding proteins (PBPs). The beta-lactam antibiotics inhibit the TP reaction, but their widespread use led to the development of drug resistance in pathogenic bacteria. In this context, the GT catalytic domain represents a potential target in the antibacterial fight. In this work, the in vitro polymerization of glycan chains by the extracellular region of recombinant Streptococcus pneumoniae PBP2a, namely, PBP2a* (the asterisk indicates the soluble form of the protein) is presented. Dansylated lipid II was used as the substrate, and the kinetic parameters K(m) and k(cat)/K(m) were measured at 40.6 micro M (+/- 15.5) and 1 x 10(-3) M(-1) s(-1), respectively. The GT reaction catalyzed by PBP2a* was inhibited by moenomycin and vancomycin. Furthermore, the sequence between Lys 78 and Ser 156 is required for enzymatic activity, whereas it is dispensable for lipid II binding. In addition, we confirmed that this region of the protein is also involved in membrane interaction, independently of the transmembrane anchor. The characterization of the catalytically active GT domain of S. pneumoniae PBP2a may contribute to the development of new inhibitors, which are urgently needed to renew the antibiotic arsenal.     

Deletion analysis of the essentiality of penicillin-binding proteins 1A, 2B and 2X of Streptococcus pneumoniae

Abstract An internal fragment from each of the penicillinebinding protein (PBP) 1A, 2B and 2X genes of Streptococcus pneumoniae , which included the region encoding the active-site serine residue, was replaced by a fragment encoding spectinomycin resistance. The resulting constructs were tested for their ability to transform S. pneumoniae strain R6 to spectinomycin resistance. Spectinomycin-resistant transformants could not be obtained using either the inactivated PBP 2X or 2B genes, suggesting that deletion of either of these genes was a lethal event, but they were readily obtained using the inactivated PBP 1A gene. Analysis using the polymerase chain reaction confirmed that the latter transformants had replaced their chromosomal copy of the PBP 1A gene with the inactivated copy of the gene. Deletion of the PBP 1A gene was therefore tolerated under laboratory conditions and appeared to have little effect on growth or susceptibility to benzylpenicillin.     

Multivariate geometrical analysis of catalytic residues in the penicillin-binding proteins

Penicillin-binding proteins (PBPs) are bacterial enzymes involved in the final stages of cell wall biosynthesis, and are targets of the β-lactam antibiotics. They can be subdivided into essential high-molecular-mass (HMM) and non-essential low-molecular-mass (LMM) PBPs, and further divided into subclasses based on sequence homologies. PBPs can catalyze transpeptidase or hydrolase (carboxypeptidase and endopeptidase) reactions. The PBPs are of interest for their role in bacterial cell wall biosynthesis, and as mechanistically interesting enzymes which can catalyze alternative reaction pathways using the same catalytic machinery. A global catalytic residue comparison seemed likely to provide insight into structure-function correlations within the PBPs. More than 90 PBP structures were aligned, and a number (40) of active site geometrical parameters extracted. This dataset was analyzed using both univariate and multivariate statistical methods. Several interesting relationships were observed. (1) Distribution of the dihedral angle for the SXXK-motif Lys side chain (DA_1) was bimodal, and strongly correlated with HMM/transpeptidase vs LMM/hydrolase classification/activity (P<0.001). This structural feature may therefore be associated with the main functional difference between the HMM and LMM PBPs. (2) The distance between the SXXK-motif Lys-NZ atom and the Lys/His-nitrogen atom of the (K/H)T(S)G-motif was highly conserved, suggesting importance for PBP function, and a possibly conserved role in the catalytic mechanism of the PBPs. (3) Principal components-based cluster analysis revealed several distinct clusters, with the HMM Class A and B, LMM Class C, and LMM Class A K15 PBPs forming one "Main" cluster, and demonstrating a globally similar arrangement of catalytic residues within this group.