Structural characterization of lytic transglycosylase MltD of Pseudomonas aeruginosa, a target for the natural product bulgecin A.

Natural product bulgecin A potentiates the activity of ß-lactam antibiotics by inhibition of three lytic transglycosylases in Pseudomonas aeruginosa, of which MltD is one. MltD exhibits both endolytic and exolytic reactions in the turnover of the cell-wall peptidoglycan and tolerates the presence or absence of stem peptides in its substrates. The present study reveals structural features of the multimodular MltD, presenting a catalytic module and four cell-wall-binding LysM modules that account for these attributes. Three X-ray structures are reported herein for MltD that disclose one unpredicted LysM module tightly attached to the catalytic domain, whereas the other LysM modules are mobile, and connected to the catalytic domain through long flexible linkers. The formation of crystals depended on the presence of bulgecin A. The expansive active-site cleft is highlighted by the insertion of a helical region, a hallmark of the family 1D of lytic transglycosylases, which was mapped out in a ternary complex of MltD:bulgecinA:chitotetraose, revealing at the minimum the presence of eight subsites (from -4 to +4, with the seat of reaction at subsites -1 and + 1) for binding of sugars of the substrate for the endolytic reaction. The mechanism of the exolytic reaction is revealed in one of the structures, showing how the substrate's terminal anhydro-NAM moiety could be sequestered at subsite +2. Our results provide the structural insight for both the endolytic and exolytic activities of MltD during cell-wall-turnover events.

DipM controls multiple autolysins and mediates a regulatory feedback loop promoting cell constriction in Caulobacter crescentus.

Proteins with a catalytically inactive LytM-type endopeptidase domain are important regulators of cell wall-degrading enzymes in bacteria. Here, we study their representative DipM, a factor promoting cell division in Caulobacter crescentus. We show that the LytM domain of DipM interacts with multiple autolysins, including the soluble lytic transglycosylases SdpA and SdpB, the amidase AmiC and the putative carboxypeptidase CrbA, and stimulates the activities of SdpA and AmiC. Its crystal structure reveals a conserved groove, which is predicted to represent the docking site for autolysins by modeling studies. Mutations in this groove indeed abolish the function of DipM in vivo and its interaction with AmiC and SdpA in vitro. Notably, DipM and its targets SdpA and SdpB stimulate each other's recruitment to midcell, establishing a self-reinforcing cycle that gradually increases autolytic activity as cytokinesis progresses. DipM thus coordinates different peptidoglycan-remodeling pathways to ensure proper cell constriction and daughter cell separation.

Crystal Structure and Pathophysiological Role of the Pneumococcal Nucleoside-binding Protein PnrA.

Nucleotides are important for RNA and DNA synthesis and, despite a de novo synthesis by bacteria, uptake systems are crucial. Streptococcus pneumoniae, a facultative human pathogen, produces a surface-exposed nucleoside-binding protein, PnrA, as part of an ABC transporter system. Here we demonstrate the binding affinity of PnrA to nucleosides adenosine, guanosine, cytidine, thymidine and uridine by microscale thermophoresis and indicate the consumption of adenosine and guanosine by 1H NMR spectroscopy. In a series of five crystal structures we revealed the PnrA structure and provide insights into how PnrA can bind purine and pyrimidine ribonucleosides but with preference for purine ribonucleosides. Crystal structures of PnrA:nucleoside complexes unveil a clear pattern of interactions in which both the N- and C- domains of PnrA contribute. The ribose moiety is strongly recognized through a conserved network of H-bond interactions, while plasticity in loop 27-36 is essential to bind purine- or pyrimidine-based nucleosides. Further, we deciphered the role of PnrA in pneumococcal fitness in infection experiments. Phagocytosis experiments did not show a clear difference in phagocytosis between PnrA-deficient and wild-type pneumococci. In the acute pneumonia infection model the deficiency of PnrA attenuated moderately virulence of the mutant, which is indicated by a delay in the development of severe lung infections. Importantly, we confirmed the loss of fitness in co-infections, where the wild-type out-competed the pnrA-mutant. In conclusion, we present the PnrA structure in complex with individual nucleosides and show that the consumption of adenosine and guanosine under infection conditions is required for virulence.

Catalytic Cycle of Glycoside Hydrolase BglX from Pseudomonas aeruginosa and Its Implications for Biofilm Formation.

BglX is a heretofore uncharacterized periplasmic glycoside hydrolase (GH) of the human pathogen Pseudomonas aeruginosa. X-ray analysis identifies it as a protein homodimer. The two active sites of the homodimer comprise catalytic residues provided by each monomer. This arrangement is seen in <2% of the hydrolases of known structure. In vitro substrate profiling shows BglX is a catalyst for ß-(1→2) and ß-(1→3) saccharide hydrolysis. Saccharides with ß-(1→4) or ß-(1→6) bonds, and the ß-(1→4) muropeptides from the cell-wall peptidoglycan, are not substrates. Additional structural insights from X-ray analysis (including structures of a mutant enzyme-derived Michaelis complex, two transition-state mimetics, and two enzyme-product complexes) enabled the comprehensive description of BglX catalysis. The half-chair (4H3) conformation of the transition-state oxocarbenium species, the approach of the hydrolytic water molecule to the oxocarbenium species, and the stepwise release of the two reaction products were also visualized. The substrate pattern for BglX aligns with the [ß-(1→2)-Glc]x and [ß-(1→3)-Glc]x periplasmic osmoregulated periplasmic glucans, and possibly with the Psl exopolysaccharides, of P. aeruginosa. Both polysaccharides are implicated in biofilm formation. Accordingly, we show that inactivation of the bglX gene of P. aeruginosa PAO1 attenuates biofilm formation.

A Structural Dissection of the Active Site of the Lytic Transglycosylase MltE from Escherichia coli.

Lytic transglycosylases (LTs) are bacterial enzymes that catalyze the cleavage of the glycan strands of the bacterial cell wall. The mechanism of this cleavage is a remarkable intramolecular transacetalization reaction, accomplished by an ensemble of active-site residues. Because the LT reaction occurs in parallel with the cell wall bond-forming reactions catalyzed by the penicillin-binding proteins, simultaneous inhibition of both enzymes can be particularly bactericidal to Gram-negative bacteria. The MltE lytic transglycosylase is the smallest of the eight LTs encoded by the Escherichia coli genome. Prior crystallographic and computational studies identified four active-site residues-E64, S73, S75, and Y192-as playing roles in catalysis. Each of these four residues was individually altered by mutation to give four variant enzymes (E64Q, S73A, S75A, and Y192F). All four variants showed reduced catalytic activity [soluble wild type (100%) > soluble Y192F and S75A (both 40%) > S73A (4%) > E64Q (≤1%)]. The crystal structure of each variant protein was determined at the resolution of 2.12 Å for E64Q, 2.33 Å for Y192F, 1.38 Å for S73A, and 1.35 Å for S75A. These variants show alteration of the hydrogen-bond interactions of the active site. Within the framework of a prior computational study of the LT mechanism, we suggest the mechanistic role of these four active-site residues in MltE catalysis.

Exolytic and endolytic turnover of peptidoglycan by lytic transglycosylase Slt of Pseudomonas aeruginosa.

ß-Lactam antibiotics inhibit cell-wall transpeptidases, preventing the peptidoglycan, the major constituent of the bacterial cell wall, from cross-linking. This causes accumulation of long non-cross-linked strands of peptidoglycan, which leads to bacterial death. Pseudomonas aeruginosa, a nefarious bacterial pathogen, attempts to repair this aberrantly formed peptidoglycan by the function of the lytic transglycosylase Slt. We document in this report that Slt turns over the peptidoglycan by both exolytic and endolytic reactions, which cause glycosidic bond scission from a terminus or in the middle of the peptidoglycan, respectively. These reactions were characterized with complex synthetic peptidoglycan fragments that ranged in size from tetrasaccharides to octasaccharides. The X-ray structure of the wild-type apo Slt revealed it to be a doughnut-shaped protein. In a series of six additional X-ray crystal structures, we provide insights with authentic substrates into how Slt is enabled for catalysis for both the endolytic and exolytic reactions. The substrate for the exolytic reaction binds Slt in a canonical arrangement and reveals how both the glycan chain and the peptide stems are recognized by the Slt. We document that the apo enzyme does not have a fully formed active site for the endolytic reaction. However, binding of the peptidoglycan at the existing subsites within the catalytic domain causes a conformational change in the protein that assembles the surface for binding of a more expansive peptidoglycan between the catalytic domain and an adjacent domain. The complexes of Slt with synthetic peptidoglycan substrates provide an unprecedented snapshot of the endolytic reaction.

Allostery, Recognition of Nascent Peptidoglycan, and Cross-linking of the Cell Wall by the Essential Penicillin-Binding Protein 2x of Streptococcus pneumoniae.

Transpeptidases, members of the penicillin-binding protein (PBP) families, catalyze cross-linking of the bacterial cell wall. This transformation is critical for the survival of bacteria, and it is the target of inhibition by ß-lactam antibiotics. We report herein our structural insights into catalysis by the essential PBP2x of Streptococcus pneumoniae by disclosing a total of four X-ray structures, two computational models based on the crystal structures, and molecular-dynamics simulations. The X-ray structures are for the apo PBP2x, the enzyme modified covalently in the active site by oxacillin (a penicillin antibiotic), the enzyme modified by oxacillin in the presence of a synthetic tetrasaccharide surrogate for the cell-wall peptidoglycan, and a noncovalent complex of cefepime (a cephalosporin antibiotic) bound to the active site. A prerequisite for catalysis by transpeptidases, including PBP2x, is the molecular recognition of nascent peptidoglycan strands, which harbor pentapeptide stems. We disclose that the recognition of nascent peptidoglycan by PBP2x takes place by complexation of one pentapeptide stem at an allosteric site located in the PASTA domains of this enzyme. This binding predisposes the third pentapeptide stem in the same nascent peptidoglycan strand to penetration into the active site for the turnover events. The complexation of the two pentapeptide stems in the same peptidoglycan strand is a recognition motif for the nascent peptidoglycan, critical for the cell-wall cross-linking reaction.

Conformational Dynamics in Penicillin-Binding Protein 2a of Methicillin-Resistant Staphylococcus aureus, Allosteric Communication Network and Enablement of Catalysis.

The mechanism of the ß-lactam antibacterials is the functionally irreversible acylation of the enzymes that catalyze the cross-linking steps in the biosynthesis of their peptidoglycan cell wall. The Gram-positive pathogen Staphylococcus aureus uses one primary resistance mechanism. An enzyme, called penicillin-binding protein 2a (PBP2a), is brought into this biosynthetic pathway to complete the cross-linking. PBP2a effectively discriminates against the ß-lactam antibiotics as potential inhibitors, and in favor of the peptidoglycan substrate. The basis for this discrimination is an allosteric site, distal from the active site, that when properly occupied concomitantly opens the gatekeeper residues within the active site and realigns the conformation of key residues to permit catalysis. We address the molecular basis of this regulation using crystallographic studies augmented by computational analyses. The crystal structures of three ß-lactams (oxacillin, cefepime, ceftazidime) complexes with PBP2a-each with the ß-lactam in the allosteric site-defined (with preceding PBP2a structures) as the "open" or "partially open" PBP2a states. A particular loop motion adjacent to the active site is identified as the driving force for the active-site conformational change that accompanies active-site opening. Correlation of this loop motion to effector binding at the allosteric site, in order to identify the signaling pathway, was accomplished computationally in reference to the known "closed" apo-PBP2a X-ray crystal structure state. This correlation enabled the computational simulation of the structures coinciding with initial peptidoglycan substrate binding to PBP2a, acyl enzyme formation, and acyl transfer to a second peptidoglycan substrate to attain cross-linking. These studies offer important insights into the structural bases for allosteric site-to-active site communication and for ß-lactam mimicry of the peptidoglycan substrates, as foundational to the mechanistic understanding of emerging PBP2a resistance mutations.