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The soluble lytic transglycosylase Cj0843c from Campylobacter jejuni breaks down cell-wall peptidoglycan (PG). Its nonhydrolytic activity sustains cell-wall remodeling and repair. We report herein our structure-function studies probing the substrate preferences and recognition by this enzyme. Our studies show that Cj0843c exhibits both exolytic and endolytic activities and forms the N-acetyl-1,6-anhydromuramyl (anhMurNAc) peptidoglycan termini, the typical transformation catalyzed by lytic transglycosylase. Cj0843c shows a trend toward a preference for substrates with anhMurNAc ends and those with peptide stems. Mutagenesis revealed that the catalytic E390 is critical for activity. In addition, mutagenesis showed that R388 and K505, located in the positively charged pocket near E390, also serve important roles. Mutation of R326, on the opposite side of this positively charged pocket, enhanced activity. Our data point to different roles for positively charged residues in this pocket for productive binding of the predominantly negatively charged PG. We also show by X-ray crystallography and by molecular dynamics simulations that the active site of Cj0843c is still capable of binding GlcNAc containing di- and trisaccharides without MurNAc moieties, without peptide stems, and without the anhMurNAc ends.

Graphical abstract, Highlights • X-ray crystallography, SAXS and MD simulations elucidates the 3D structure for saPBP1. • 3D structure of saPBP1 reveals dynamical mobility for the pedestal and PASTA domains. • Structures with β-lactams and penta-Gly provide model for peptidoglycan binding. • Mass-spectrometry reveals peptidoglycan binding by PASTA and the non-active-site regions. • Computation provides a dynamic model for full-length PBP1 and its complex with FtsW., The penicillin-binding proteins are the enzyme catalysts of the critical transpeptidation crosslinking polymerization reaction of bacterial peptidoglycan synthesis and the molecular targets of the penicillin antibiotics. Here, we report a combined crystallographic, small-angle X-ray scattering (SAXS) in-solution structure, computational and biophysical analysis of PBP1 of Staphylococcus aureus (saPBP1), providing mechanistic clues about its function and regulation during cell division. The structure reveals the pedestal domain, the transpeptidase domain, and most of the linker connecting to the “penicillin-binding protein and serine/threonine kinase associated” (PASTA) domains, but not its two PASTA domains, despite their presence in the construct. To address this absence, the structure of the PASTA domains was determined at 1.5 Å resolution. Extensive molecular-dynamics simulations interpret the PASTA domains of saPBP1 as conformationally mobile and separated from the transpeptidase domain. This conclusion was confirmed by SAXS experiments on the full-length protein in solution. A series of crystallographic complexes with β-lactam antibiotics (as inhibitors) and penta-Gly (as a substrate mimetic) allowed the molecular characterization of both inhibition by antibiotics and binding for the donor and acceptor peptidoglycan strands. Mass-spectrometry experiments with synthetic peptidoglycan fragments revealed binding by PASTA domains in coordination with the remaining domains. The observed mobility of the PASTA domain in saPBP1 could play a crucial role for in vivo interaction with its glycosyltransferase partner in the membrane or with other components of the divisome machinery, as well as for coordination of transpeptidation and polymerization processes in the bacterial divisome.

8 pags., 5 figs., 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, The work at the University of Notre Dame was supported by grants from the National Institutes of Health (GM61629 and GM131685), and that in Spain by a grant from MICIU Ministry (BFU2017-90030-P). The authors thank the staff from the ALBA (Barcelona, Spain) synchrotron facility for help in X-ray data collection and CRC of the University of Notre Dame for the computing resources. The authors acknowledge Grant P30 DK089507 from the National Institutes of Health for the BglX transposon mutant of P. aeruginosa.

The soluble lytic transglycosylase Cj0843c from Campylobacter jejuni breaks down cell-wall peptidoglycan (PG). Its non-hydrolytic activity sustains cell-wall remodeling and repair. We report herein our structure-function studies probing the substrate preferences and recognition by this enzyme. Our studies show that Cj0843c exhibits both exolytic and endolytic activities and forms the N-acetyl-1,6-anhydromuramyl (anhMurNAc) peptidoglycan termini, the typical transformation catalyzed by lytic transglycosylase. Cj0843c shows a trend toward a preference of substrates with anhMurNAc ends and those with peptide stems. Mutagenesis revealed that the catalytic E390 is critical for activity. In addition, mutagenesis showed that R388 and K505, located in the positively charged pocket near E390, also serve important roles. Mutation of R326, on the opposite side of this positively charged pocket, enhanced activity. Our data point to different roles for positively charged residues in this pocket for productive binding of the predominantly negatively charged PG. We also show by X-ray crystallography and by MD simulations that the active site of Cj0843c is still capable of binding GlcNAc containing di- and trisaccharides without MurNAc moieties, without peptide stems, and without the anhMurNAc ends.

[Image: see text] Diabetic foot ulcers (DFUs) are a common complication of diabetes that are recalcitrant to healing due to persistent inflammation. The majority of DFUs have bacterial biofilms, with Staphylococcus epidermidis as a predominant bacterium, requiring infection control with antibiotics before treatment of the wound. Matrix metalloproteinases (MMPs) play roles in the pathology and repair of DFUs. However, defining the roles of the 24 human MMPs has been challenging due to the presence of three forms for each MMP, of which only one is catalytically competent, and the lack of convenient methods to distinguish among the three forms of MMPs. Using an affinity resin that binds only to the active forms of MMPs, with identification and quantification by mass spectrometry, we found that infected wounds in mice had increased levels of active MMP-9 compared to uninfected ones, paralleling infected human DFUs. MMP-9 activity prevents diabetic wounds from healing. We evaluated the efficacy of the selective small-molecule MMP-9 inhibitor, (R)-ND-336, in the infected diabetic mouse model of wound healing and showed that (R)-ND-336 alone or in combination with the antibiotic linezolid improves wound healing by inhibiting the detrimental MMP-9, mitigating macrophage infiltration to diminish inflammation, and increasing angiogenesis to restore the normal wound healing process. An advantage of this strategy is the ability to administer (R)-ND-336 concurrently with an antibiotic.

Chronic wounds are a complication of diabetes. Treatment for diabetic foot ulcers is complex with little clinical recourse, resulting in 108,000 lower-limb amputations annually in the United States alone. Matrix metalloproteinases (MMPs) play important roles in the pathology and in the repair of chronic wounds. We previously identified active MMP-8 and MMP-9 in wounds of diabetic mice and determined that MMP-8 accelerates wound repair, while MMP-9 is the culprit for the diabetic wound being refractory to healing. Aclerastide, a peptide analog of angiotensin II, recently failed in phase III clinical trials for treatment of diabetic foot ulcers. We demonstrate herein that treatment of wounds of diabetic mice with aclerastide results in elevated levels of reactive oxygen species and of active MMP-9, which is likely an important contributor to the failure of aclerastide in clinical trials.

The major constituent of bacterial cell wall is peptidoglycan, which in its crosslinked form is a polymer of considerable complexity that encases the entire bacterium. A functional cell wall is indispensable for the survival of the organism. There are several dozens of enzymes that assemble and disassemble the peptidoglycan dynamically within each bacterial generation. Understanding of the nature of these transformations is critical knowledge on these events. Octasaccharide peptidoglycans were prepared and studied with seven recombinant cell-wall-active enzymes (SltB1, MltB, RlpA, mutanolysin, AmpDh2, AmpDh3 and PBP5). With the use of highly sensitive mass spectrometry methods, we describe the breadth of reactions that these enzymes catalyze with the peptidoglycan and shed light on the nature of the cell-wall alteration performed by these enzymes. The enzymes exhibit broadly distinct preferences for their substrate peptidoglycans in the reactions that they catalyze.

Dimethylmaleoyl (DMM) moiety has become an important amine protective group in sugar chemistry. We disclose herein that DMM-containing D-glucosamine analogues, because of their electrophilic nature, are prone to reactions with strong nucleophiles, such as hydrazine, resulting in a set of undesired side products that are difficult to detect, yet proved to be problematic for organic synthesis.

Diabetic foot ulcers are characterized by hypoxia. For many patients, hyperbaric oxygen (HBO) therapy is the last recourse for saving the limb from amputation, for which the molecular basis is not understood. We previously identified the active form of matrix metalloproteinase-9 (MMP-9) as responsible for diabetic foot ulcer's recalcitrance to healing. Transcription of mmp-9 to the inactive zymogen is upregulated during hypoxia. Activation of the zymogen is promoted by proteases and reactive oxygen species (ROS). We hypothesized that the dynamics of these two events might lead to a lowering of active MMP-9 levels in the wounded tissue. We employed the full-thickness excisional db/db mouse model to study wound healing, and treated the mice to 3.0 atm of molecular oxygen for 90 minutes, 5 days per week for 10 days in an HBO research chamber. Treatment with HBO accelerated diabetic wound healing compared to untreated mice, with more completed and extended reepithelialization. We imaged the wounds for ROS in vivo with a luminol-based probe and found that HBO treatment actually decreases ROS levels. The levels of superoxide dismutase, catalase, and glutathione peroxidase-enzymes that turn over ROS-increased after HBO treatment, hence the observation of decreased ROS. Since ROS levels are lowered, we explored the effect that this would have on activation of MMP-9. Quantitative analysis with an affinity resin that binds and pulls down the active MMPs exclusively, coupled with proteomics, revealed that HBO treatment indeed reduces the active MMP-9 levels. This work for the first time demonstrates that diminution of active MMP-9 is a contributing factor and a mechanism for enhancement of diabetic wound healing by HBO therapy.

An enzyme superfamily, the lytic transglycosylases (LTs), occupies the space between the two membranes of Gram-negative bacteria. LTs catalyze the non-hydrolytic cleavage of the bacterial peptidoglycan cell-wall polymer. This reaction is central to the growth of the cell wall, for excavating the cell wall for protein insertion, and for monitoring the cell wall so as to initiate resistance responses to cell-wall-acting antibiotics. The nefarious Gram-negative pathogen Pseudomonas aeruginosa encodes eleven LTs. With few exceptions, their substrates and functions are unknown. Each P. aeruginosa LT was expressed as a soluble protein and evaluated with a panel of substrates (both simple and complex mimetics of their natural substrates). Thirty-one distinct products distinguish these LTs with respect to substrate recognition, catalytic activity, and relative exolytic or endolytic ability. These properties are foundational to an understanding of the LTs as catalysts and as antibiotic targets.

The repeating isoprene unit is a fundamental biosynthetic motif. The repetitive structure presents challenges both for synthesis and for structural characterization. In this synthesis of the (Z8,E2,ω)-undecaprenol of prokaryotic glycobiology, we exemplify solutions to these challenges. Allylation of sulfone-derived carbanions controlled the stereochemistry, and its proof-of-structure was secured by Eu(hfc)3 complexation to disperse the overlaid resonances of its 1H NMR spectrum.

Neisseria gonorrhoeae releases peptidoglycan (PG) fragments during infection that provoke a large inflammatory response and, in pelvic inflammatory disease, this response leads to the death and sloughing of ciliated cells of the Fallopian tube. We characterized the biochemical functions and localization of two enzymes responsible for the release of proinflammatory PG fragments. The putative lytic transglycosylases LtgA and LtgD were shown to create the 1,6-anhydromuramyl moieties, and both enzymes were able to digest a small, synthetic tetrasaccharide dipeptide PG fragment into the cognate 1,6-anhydromuramyl-containing reaction products. Degradation of tetrasaccharide PG fragments by LtgA is the first demonstration of a family 1 lytic transglycosylase exhibiting this activity. Pulse-chase experiments in gonococci demonstrated that LtgA produces a larger amount of PG fragments than LtgD, and a vast majority of these fragments are recycled. In contrast, LtgD was necessary for wild-type levels of PG precursor incorporation and produced fragments predominantly released from the cell. Additionally, super-resolution microscopy established that LtgA localizes to the septum, whereas LtgD is localized around the cell. This investigation suggests a model where LtgD produces PG monomers in such a way that these fragments are released, whereas LtgA creates fragments that are mostly taken into the cytoplasm for recycling.

Summary The pneumococcal autolysin LytA is a key virulence factor involved in several important functions including DNA competence, immune evasion and biofilm formation. Here, we present the 1.05 A crystal structure of the catalytic domain of LytA in complex with a synthetic cell-wall-based peptidoglycan (PG) ligand that occupies the entire Y-shaped substrate-binding crevice. As many as twenty-one amino-acid residues are engaged in ligand interactions with a majority of these interactions directed towards the glycan strand. All saccharides are intimately bound through hydrogen bond, van der Waals and CH-π interactions. Importantly, the structure of LytA is not altered upon ligand binding, whereas the bound ligand assumes a different conformation compared to the unbound NMR-based solution structure of the same PG-fragment. Mutational study reveals that several non-catalytic glycan-interacting residues, structurally conserved in other amidases from Gram-positive Firmicutes, are pivotal for enzymatic activity. The three-dimensional structure of the LytA/PG complex provides a novel structural basis for ligand restriction by the pneumococcal autolysin, revealing for the first time an importance of the multivalent binding to PG saccharides.

Muropeptides are a group of bacterial natural products generated from the cell wall in the course of its turnover. These compounds are cell-wall recycling intermediates and are also involved in signaling within the bacterium. However, the identity of these signaling molecules remains elusive. The identification and characterization of 20 muropeptides from Pseudomonas aeruginosa is described. The least abundant of these metabolites is present at 100 and the most abundant at 55,000 molecules per bacterium. Analysis of these muropeptides under conditions of induction of resistance to a β-lactam antibiotic identified two signaling muropeptides (N-acetylglucosamine-1,6-anhydro-N-acetylmuramyl pentapeptide and 1,6-anhydro-N-acetylmuramyl pentapeptide). Authentic synthetic samples of these metabolites were shown to activate expression of β-lactamase in the absence of any β-lactam antibiotic, thus indicating that they serve as chemical signals in this complex biochemical pathway.

The human pathogen Pseudomonas aeruginosa harbors three paralogous zinc proteases annotated as AmpD, AmpDh2, and AmpDh3, which turn over the cell wall and cell-wall-derived muropeptides. AmpD is cytoplasmic and plays a role in recycling of cell-wall muropeptides, with a link to antibiotic resistance. AmpDh2 and AmpDh3 are periplasmic enzymes; with the former anchored to the inner leaflet of the outer membrane and the latter a soluble enzyme. We document herein that the type VI secretion system locus II (H2-T6SS) of P. aeruginosa delivers AmpDh3 (but not AmpD or AmpDh2) to the periplasm of a prey bacterium upon contact. AmpDh3 hydrolyzes the cell-wall peptidoglycan of the prey bacterium, which leads to its killing, thereby providing a growth advantage for P. aerugionsa in bacterial competition. We also document that the periplasmic protein PA0808, heretofore of unknown function, affords self-protection from lysis by AmpDh3. Cognates of the AmpDh3-PA0808 pair are widely distributed across Gram-negative bacteria, underscoring the importance of their function as an evolutionary advantage and that of the H2-T6SS as the means for manifestation of the effect.

Diabetic foot ulcers (DFUs) are a significant health problem. A single existing FDA-approved drug for this ailment, becaplermin, is not standard-of-care. We previously demonstrated that upregulation of active matrix metalloproteinase (MMP)-9 is the reason that the diabetic wound in mice is recalcitrant to healing and that MMP-8 participates in wound repair. In the present study, we validate the target MMP-9 by identifying and quantifying active MMP-8 and MMP-9 in human diabetic wounds using an affinity resin that binds exclusively to the active forms of MMPs coupled with proteomics. Furthermore, we synthesize and evaluate enantiomerically pure ( R)- and ( S)-ND-336, as inhibitors of the detrimental MMP-9, and show that the ( R)-enantiomer has superior efficacy in wound healing over becaplermin. Our results reveal that the mechanisms of pathology and repair are similar in diabetic mice and diabetic humans and that ( R)-ND-336 holds promise for the treatment of DFUs as a first-in-class therapeutic.

β-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., The work in the United States was supported by NIH Grant GM61629 (to S.M.), and in Madrid, Spain, by Spanish Ministry of Economy and Competitiveness Grants BFU2014-59389-P and BFU2017-90030-P (to J.A.H.). D.A.D. is a Fellow of the Chemistry–Biochemistry–Biology Interface Program (NIH Training Grant T32GM075762) and a Fellow of the Eck Institute of Global Health at the University of Notre Dame. Grants BIO2015-64216-P (to C.M.), MDM2014- 0435-01 (to I.U.), and BES-2015-071397 scholarship (to C.M.) supported the work in Barcelona, Spain.

β-Lactam antibiotics are currently the most broadly used class of antibiotics. These antibiotics prevent bacterial cell wall from cross-linking, which leads to the accumulation of long non–cross-linked strands of peptidoglycan. Pseudomonas aeruginosa, a nefarious bacterial pathogen, attempts to repair this aberrantly formed peptidoglycan by the function of the lytic transglycosylase Slt. We document in the present report that Slt turns over the peptidoglycan by both scission of the glycosidic bonds from a terminus or in the middle of the peptidoglycan. In a series of seven X-ray crystal structures, we provide structural context to how these two reactions take place. These results disclose the details of bacterial response to the β-lactam antibiotic challenge.

Background: Csd6 is one of the cell shape-determining proteins in H. pylori. Results: The active site of Csd6 is tailored to function as an l,d-carboxypeptidase in the peptidoglycan-trimming process. Conclusion: Csd6 constitutes a new family of l,d-carboxypeptidase. Significance: The substrate limitation of Csd6 is a strategy that H. pylori uses to regulate its helical cell shape and motility., Helicobacter pylori causes gastrointestinal diseases, including gastric cancer. Its high motility in the viscous gastric mucosa facilitates colonization of the human stomach and depends on the helical cell shape and the flagella. In H. pylori, Csd6 is one of the cell shape-determining proteins that play key roles in alteration of cross-linking or by trimming of peptidoglycan muropeptides. Csd6 is also involved in deglycosylation of the flagellar protein FlaA. To better understand its function, biochemical, biophysical, and structural characterizations were carried out. We show that Csd6 has a three-domain architecture and exists as a dimer in solution. The N-terminal domain plays a key role in dimerization. The middle catalytic domain resembles those of l,d-transpeptidases, but its pocket-shaped active site is uniquely defined by the four loops I to IV, among which loops I and III show the most distinct variations from the known l,d-transpeptidases. Mass analyses confirm that Csd6 functions only as an l,d-carboxypeptidase and not as an l,d-transpeptidase. The d-Ala-complexed structure suggests possible binding modes of both the substrate and product to the catalytic domain. The C-terminal nuclear transport factor 2-like domain possesses a deep pocket for possible binding of pseudaminic acid, and in silico docking supports its role in deglycosylation of flagellin. On the basis of these findings, it is proposed that H. pylori Csd6 and its homologs constitute a new family of l,d-carboxypeptidase. This work provides insights into the function of Csd6 in regulating the helical cell shape and motility of H. pylori.

SB-3CT is a potent and selective inhibitor of matrix metalloproteinase (MMP)-2 and -9, which has shown efficacy in an animal model of severe traumatic brain injury (TBI). However, SB-3CT is poorly water-soluble and is metabolized primarily to p-hydroxy SB-3CT (2), a more potent inhibitor than SB-3CT. We synthesized the O-phosphate prodrug (3) of compound 2 to enhance its water solubility by more than 2000-fold. The prodrug 3 was a poor MMP inhibitor, but readily hydrolyzed to the active 2 in human blood. Pharmacokinetics and brain distribution studies in mice showed that 2 crossed the blood-brain barrier (BBB) and achieved therapeutic concentrations in the brain. The prodrug 3/compound 2 was evaluated in a mouse model of severe TBI and found to significantly decrease the brain lesion volume and improve neurological outcomes. MMP-9 inhibition by a water-soluble thiirane inhibitor is a promising therapy for treatment of TBI.

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 β-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.

Farnesol, a sesquiterpene alcohol, potentiates the activity of β-lactam antibiotics against antibiotic-resistant bacteria. We document that farnesol and two synthetic derivatives (compounds 2 and 6) have poor antibacterial activities of their own, but they potentiate the activities of ampicillin and oxacillin against Staphylococcus aureus strains (including methicillin-resistant S. aureus). These compounds attenuate the rate of growth of bacteria, which has to be taken into account in assessment of the potentiation effect.

Pseudomonas aeruginosa is an opportunistic Gram-negative bacterial pathogen. A primary contributor to its ability to resist β-lactam antibiotics is the expression, following detection of the β-lactam, of the AmpC β-lactamase. As AmpC expression is directly linked to the recycling of the peptidoglycan of the bacterial cell wall, an important question is the identity of the signaling molecule(s) in this relationship. One mechanism used by clinical strains to elevate AmpC expression is loss of function of penicillin-binding protein 4 (PBP4). As the mechanism of the β-lactams is PBP inactivation, this result implies that the loss of the catalytic function of PBP4 ultimately leads to induction of antibiotic resistance. PBP4 is a bifunctional enzyme having both dd-carboxypeptidase and endopeptidase activities. Substrates for both the dd-carboxypeptidase and the 4,3-endopeptidase activities were prepared by multistep synthesis, and their turnover competence with respect to PBP4 was evaluated. The endopeptidase activity is specific to hydrolysis of 4,3-cross-linked peptidoglycan. PBP4 catalyzes both reactions equally well. When P. aeruginosa is grown in the presence of a strong inducer of AmpC, the quantities of both the stem pentapeptide (the substrate for the dd-carboxypeptidase activity) and the 4,3-cross-linked peptidoglycan (the substrate for the 4,3-endopeptidase activity) increase. In the presence of β-lactam antibiotics these altered cell-wall segments enter into the muropeptide recycling pathway, the conduit connecting the sensing event in the periplasm and the unleashing of resistance mechanisms in the cytoplasm.

H. pylori Csd4 (HP1075), together with other peptidoglycan hydrolases, plays an important role in determining the helical cell shape. Its crystal structure has been determined in three different forms., Helicobacter pylori infection causes a variety of gastrointestinal diseases, including peptic ulcers and gastric cancer. Its colonization of the gastric mucosa of the human stomach is a prerequisite for survival in the stomach. Colonization depends on its motility, which is facilitated by the helical shape of the bacterium. In H. pylori, cross-linking relaxation or trimming of peptidoglycan muropeptides affects the helical cell shape. Csd4 has been identified as one of the cell shape-determining peptidoglycan hydrolases in H. pylori. It is a Zn2+-dependent d,l-carboxypeptidase that cleaves the bond between the γ-d-Glu and the mDAP of the non-cross-linked muramyl­tripeptide (muramyl-l-Ala-γ-d-Glu-mDAP) of the peptidoglycan to produce the muramyldipeptide (muramyl-l-Ala-γ-d-Glu) and mDAP. Here, the crystal structure of H. pylori Csd4 (HP1075 in strain 26695) is reported in three different states: the ligand-unbound form, the substrate-bound form and the product-bound form. H. pylori Csd4 consists of three domains: an N-terminal d,l-carboxypeptidase domain with a typical carboxy­peptidase fold, a central β-barrel domain with a novel fold and a C-terminal immunoglobulin-like domain. The d,l-carboxypeptidase domain recognizes the substrate by interacting primarily with the terminal mDAP moiety of the muramyltripeptide. It undergoes a significant structural change upon binding either mDAP or the mDAP-containing muramyl­tripeptide. It it also shown that Csd5, another cell-shape determinant in H. pylori, is capable of interacting not only with H. pylori Csd4 but also with the dipeptide product of the reaction catalyzed by Csd4.

The compound 2-[(4-phenoxyphenyl)sulfonylmethyl]thiirane, C15H14O3S2, a selective gelatinase inhibitor, was synthesized and structurally characterized. Two crystals were analyzed, one each for theRandSenantiomers, and the results were compared with the previously reported structure of the racemate. The enantiomerically pure compounds both crystallize withZ′ = 2 in the space groupP21, while the racemic mixture crystallizes withZ′ = 1 in the space groupP21/c, with disorder in the position of the thiirane group. This disorder accommodates both molecules for each of the enantiomerically pure crystals, showing good overlap of the molecules of the pure enantiomorphs with the components of the centrosymmetric structure.

Bacterial cell wall is a polymer of considerable complexity that is in constant equilibrium between synthesis and recycling. AmpDh3 is a periplasmic zinc protease of Pseudomonas aeruginosa, which is intimately involved in cell-wall remodeling. We document the hydrolytic reactions that this enzyme performs on the cell wall. The process removes the peptide stems from the peptidoglycan, the major constituent of the cell wall. We document that the majority of the reactions of this enzyme takes place on the polymeric insoluble portion of the cell wall, as opposed to the fraction that is released from it. We show that AmpDh3 is tetrameric both in crystals and in solution. Based on the X-ray structures of the enzyme in complex with two synthetic cell-wall-based ligands, we present for the first time a model for a multivalent anchoring of AmpDh3 onto the cell wall, which lends itself to its processive remodeling. © 2013 American Chemical Society.

The N-acetylglucosaminidase NagZ of Pseudomonas aeruginosa catalyzes the first cytoplasmic step in recycling of muropeptides, cell-wall-derived natural products. This reaction regulates gene expression for the β-lactam resistance enzyme, β-lactamase. The enzyme catalyzes hydrolysis of N-acetyl-β-d-glucosamine-(1→4)-1,6-anhydro-N-acetyl-β-d-muramyl-peptide (1) to N-acetyl-β-d-glucosamine (2) and 1,6-anhydro-N-acetyl-β-d-muramyl-peptide (3). The structural and functional aspects of catalysis by NagZ were investigated by a total of seven X-ray structures, three computational models based on the X-ray structures, molecular-dynamics simulations and mutagenesis. The structural insights came from the unbound state and complexes of NagZ with the substrate, products and a mimetic of the transient oxocarbenium species, which were prepared by synthesis. The mechanism involves a histidine as acid/base catalyst, which is unique for glycosidases. The turnover process utilizes covalent modification of D244, requiring two transition-state species and is regulated by coordination with a zinc ion. The analysis provides a seamless continuum for the catalytic cycle, incorporating large motions by four loops that surround the active site.

Neisseria gonorrhoeae releases peptidoglycan (PG) fragments during infection that provoke a large inflammatory response and, in pelvic inflammatory disease, this response leads to the death and sloughing of ciliated cells of the Fallopian tube. We characterized the biochemical functions and localization of two enzymes responsible for the release of proinflammatory PG fragments. The putative lytic transglycosylases LtgA and LtgD were shown to create the 1,6-anhydromuramyl moieties, and both enzymes were able to digest a small, synthetic tetrasaccharide dipeptide PG fragment into the cognate 1,6-anhydromuramyl-containing reaction products. Degradation of tetrasaccharide PG fragments by LtgA is the first demonstration of a family 1 lytic transglycosylase exhibiting this activity. Pulse-chase experiments in gonococci demonstrated that LtgA produces a larger amount of PG fragments than LtgD, and a vast majority of these fragments are recycled. In contrast, LtgD was necessary for wild-type levels of PG precursor incorporation and produced fragments predominantly released from the cell. Additionally, super-resolution microscopy established that LtgA localizes to the septum, whereas LtgD is localized around the cell. This investigation suggests a model where LtgD produces PG monomers in such a way that these fragments are released, whereas LtgA creates fragments that are mostly taken into the cytoplasm for recycling.

Despite significant structural rearrangement upon desolvation of a three-dimensional molecular framework of hexaaquacobalt cations and redox- active functionalized tetrathiafulvalene anions (see the picture; the area filled with water molecules is shown in gray), monocrystallinity and microporosity are retained. X-ray analyses show that a unique combination of hydrogen bonds and π ·· π interactions within the framework gives this material a structural flexibility not seen in zeolites or their analogues.

A novel 5,10,15,20-tetrakis(R-substituted) porphyrin receptor molecule (R = C6H4NHC(O)C5H4CoC5H5+ PF6–)3 is prepared and is shown to spectrally and electrochemically sense halide, nitrate, hydrogen sulfate and dihydrogen phosphate guest anions.

The synthesis of a novel neutral fluoride anion selective bis-calix[4]arene receptor is described in which the upper rim of one calix[4]arene moietu is linked via amide bonds to the lower rim of another. © 1995.

New upper-rim bis[rhenium(I) bipyridyl, ruthenium(II) bis(bipyridyl), cobaltocenium]calix[4]arene receptors exhibit remarkable dihydrogen phosphate selective anion recognition properties dictated by the relative positions of lower-rim tosyl substituents.

The pneumococcal autolysin LytA is a key virulence factor involved in several important functions including DNA competence, immune evasion and biofilm formation. Here, we present the 1.05 Å crystal structure of the catalytic domain of LytA in complex with a synthetic cell-wall-based peptidoglycan (PG) ligand that occupies the entire Y-shaped substrate-binding crevice. As many as twenty-one amino-acid residues are engaged in ligand interactions with a majority of these interactions directed towards the glycan strand. All saccharides are intimately bound through hydrogen bond, van der Waals and CH-π interactions. Importantly, the structure of LytA is not altered upon ligand binding, whereas the bound ligand assumes a different conformation compared to the unbound NMR-based solution structure of the same PG-fragment. Mutational study reveals that several non-catalytic glycan-interacting residues, structurally conserved in other amidases from Gram-positive Firmicutes, are pivotal for enzymatic activity. The three-dimensional structure of the LytA/PG complex provides a novel structural basis for ligand restriction by the pneumococcal autolysin, revealing for the first time an importance of the multivalent binding to PG saccharides.

Four possible isomers of essramycin, a natural product from a marine Streptomyces species isolated from the Egyptian Mediterranean coast, were synthesized. The structures for the isomers were assigned unequivocally by (1)H NMR, (13)C NMR, high-resolution mass spectrometry, and X-ray crystal structure determinations. Notwithstanding the earlier report of broad-spectrum antibacterial activity for the natural product, none of the four isomers exhibited any such activity.

A family of 11 lytic transglycosylases in Pseudomonas aeruginosa, an opportunistic human pathogen, turn over the polymeric bacterial cell wall in the course of its recycling, repair, and maturation. The functions of these enzymes are not fully understood. We disclose herein that SltB3 of P. aeruginosa is an exolytic lytic transglycosylase. We characterize its reaction and its products by the use of peptidoglycan-based molecules. The enzyme recognizes a minimum of four sugars in its substrate but can process a substrate comprised of a peptidoglycan of 20 sugars. The ultimate product of the reaction is N-acetylglucosamine-1,6-anhydro-N-acetylmuramic acid. The X-ray structure of this enzyme is reported for the first time. The enzyme is comprised of four domains, arranged within an annular conformation. The polymeric linear peptidoglycan substrate threads through the opening of the annulus, as it experiences turnover.

We describe a practical, multigram synthesis of (2Z,6Z,10Z,14Z,18E,22E)-3,7,11,15,19,23,27-heptamethyl-2,6,10,14,18,22,26-octacosaheptaen-1-ol [(Z(4),E(2),ω)-heptaprenol, 4] using the nerol-derived sulfone 8 as the key intermediate. Sulfone 8 is prepared by the literature route and is converted in five additional steps (18% yield from 8) to (Z(4),E(2),ω)-heptaprenol 4. The use of Eu(hfc)(3) as an NMR shift reagent not only enabled confirmation of the structure and stereochemistry of 4, but further enabled the structural assignment to a major side product from a failed synthetic connection. The availability by this synthesis of (Z(4),E(2),ω)-heptaprenol 4 in gram quantities will enable preparative access to key reagents for the study of the biosynthesis of the bacterial cell envelope.

Gram-negative bacteria have evolved an elaborate process for the recycling of their cell wall, which is initiated in the periplasmic space by the action of lytic transglycosylases. The product of this reaction, β-d-N-acetylglucosamine-(1→4)-1,6-anhydro-β-d-N-acetylmuramyl-l-Ala-γ-d-Glu-meso-DAP-d-Ala-d-Ala (compound 1), is internalized to begin the recycling events within the cytoplasm. The first step in the cytoplasmic recycling is catalyzed by the NagZ glycosylase, which cleaves in a hydrolytic reaction the N-acetylglucosamine glycosidic bond of metabolite 1. The reactions catalyzed by both the lytic glycosylases and NagZ are believed to involve oxocarbenium transition species. We describe herein the synthesis and evaluation of four iminosaccharides as possible mimetics of the oxocarbenium species, and we disclose one as a potent (compound 3, Ki = 300 ± 15 nM) competitive inhibitor of NagZ.

The outer membrane protein A (OmpA) plays important roles in anchoring of the outer membrane to the bacterial cell wall. The C-terminal periplasmic domain of OmpA (OmpA-like domain) associates with the peptidoglycan (PGN) layer noncovalently. However, there is a paucity of information on the structural aspects of the mechanism of PGN recognition by OmpA-like domains. To elucidate this molecular recognition process, we solved the high-resolution crystal structure of an OmpA-like domain from Acinetobacter baumannii bound to diaminopimelate (DAP), a unique bacterial amino acid from the PGN. The structure clearly illustrates that two absolutely conserved Asp271 and Arg286 residues are the key to the binding to DAP of PGN. Identification of DAP as the central anchoring site of PGN to OmpA is further supported by isothermal titration calorimetry and a pulldown assay with PGN. An NMR-based computational model for complexation between the PGN and OmpA emerged, and this model is validated by determining the crystal structure in complex with a synthetic PGN fragment. These structural data provide a detailed glimpse of how the anchoring of OmpA to the cell wall of gram-negative bacteria takes place in a DAP-dependent manner.—Park, J. S., Lee, W. C., Yeo, K. J., Ryu, K.-S., Kumarasiri, M., Hesek, D., Lee, M., Mobashery, S., Song, J. H., Lim, S. I., Lee, J. C., Cheong, C., Jeon, Y. H., Kim, H.-Y. Mechanism of anchoring of OmpA protein to the cell wall peptidoglycan of the gram-negative bacterial outer membrane.