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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.

El pdf del artículo es el manuscrito de autor (PMID:21341761)., The crystal structure of the first endolytic peptidoglycan lytic transglycosylase MltE from Escherichia coli is reported here. The degradative activity of this enzyme initiates the process of cell wall recycling, which is an integral event in the existence of bacteria. The structure sheds light on how MltE recognizes its substrate, the cell wall peptidoglycan. It also explains the ability of this endolytic enzyme to cleave in the middle of the peptidoglycan chains. Furthermore, the structure reveals how the enzyme is sequestered on the inner leaflet of the outer membrane. © 2011 American Chemical Society., Este trabajo fue finanaciado por las concesiones bfu2008-01711 y eu-cp223111.

© 2014 FEBS. Molecular replacement, one of the general methods used to solve the crystallographic phase problem, relies on the availability of suitable models for placement in the unit cell of the unknown structure in order to provide initial phases. ARCIMBOLDO, originally conceived for ab initio phasing, operates at the limit of this approach, using small, very accurate fragments such as polyalanine α-helices. A distant homolog may contain accurate building blocks, but it may not be evident which sub-structure is the most suitable purely from the degree of conservation. Trying out all alternative possibilities in a systematic way is computationally expensive, even if effective. In the present study, the solution of the previously unknown structure of MltE, an outer membrane-anchored endolytic peptidoglycan lytic transglycosylase from Escherichia coli, is described. The asymmetric unit contains a dimer of this 194 amino acid protein. The closest available homolog was the catalytic domain of Slt70 (PDB code 1QTE). Originally, this template was used omitting contiguous spans of aminoacids and setting as many ARCIMBOLDO runs as models, each aiming to locate two copies sequentially with PHASER. Fragment trimming against the correlation coefficient prior to expansion through density modification and autotracing in SHELXE was essential. Analysis of the figures of merit led to the strategy to optimize the search model against the experimental data now implemented within ARCIMBOLDO-SHREDDER (http://chango.ibmb.csic.es/SHREDDER). In this strategy, the initial template is systematically shredded, and fragments are scored against each unique solution of the rotation function. Results are combined into a score per residue and the template is trimmed accordingly., This work was supported by grants BFU2012-35367, IDC-20101173 and BFU2011-25326 (from the Spanish Ministry of Economy and Competitiveness), grant 2009SGR-1036 from the Generalitat de Catalunya (2009SGR-1036) and the Government of Community of Madrid (S2010/BMD-2457). KM thanks the Deutsche Forschungsgemeinschaft and the Spanish Ministerio de Economía y Competitividad (ME 3679/1-1)/Juan de la Cierva Sub-Program for support

4 pags, 4 figs. -- Supporting Information is available at the Publisher web., The zinc protease AmpDh2 is a virulence determinant of Pseudomonas aeruginosa, a problematic human pathogen. The mechanism of how the protease manifests virulence is not known, but it is known that it turns over the bacterial cell wall. The reaction of AmpDh2 with the cell wall was investigated, and nine distinct turnover products were characterized by LC/MS/MS. The enzyme turns over both the cross-linked and noncross-linked cell wall. Three high-resolution X-ray structures, the apo enzyme and two complexes with turnover products, were solved. The X-ray structures show how the dimeric protein interacts with the inner leaflet of the bacterial outer membrane and that the two monomers provide a more expansive surface for recognition of the cell wall. This binding surface can accommodate the 3D solution structure of the cross-linked cell wall. © 2013 American Chemical Society., This work was supported by a grant from the NIH (GM61629) and by grants BFU2011-25326 (the Spanish Ministry of Economy and Competitiveness) and S2010/BMD-2457 (the Government of Community of Madrid). The Mass Spectrometry & Proteomics Facility of the University of Notre Dame is supported by grant CHE0741793 from the NSF.

3 pags, 2 figs, 1 tab, MltE from Escherichia coli (193 amino acids, 21 380 Da) is a lytic trans-glycosylase that initiates the first step of cell-wall recycling. This enzyme is responsible for the cleavage of the cell-wall peptidoglycan at the Β-1,4-glycosidic bond between the N-acetylglucosamine and N-acetylmuramic acid units. At the end this reaction generates a disaccharide that is internalized and initiates the recycling process. To obtain insights into the biological functions of MltE, crystallization trials were performed and crystals of MltE protein that were suitable for X-ray diffraction analysis were obtained. The MltE protein of E. coli was crystallized using the hanging-drop vapour-diffusion method at 291 K. Crystals grew from a mixture consisting of 28% polyethylene glycol 4000, 0.1 M Tris pH 8.4 and 0.2 M magnesium chloride. Further optimization was performed using the microbatch technique. Single crystals were obtained that belonged to the orthorhombic space group C2221, with unit-cell parameters a = 123.32, b = 183.93, c = 35.29 Å, and diffracted to a resolution of 2.1 Å. © 2011 International Union of Crystallography. All rights reserved., This work was supported by grants from the Spanish Ministry of Science and Technology (BFU2008-01711 and the Factoría de Cristalización from CONSOLIDER-INGENIO 2010) and the COMBACT program (S-BIO-0260/2006). The work in the USA was supported by the National Institutes of Health. CA-R is a fellow of the Spanish Ministry of Education and Science (BFU2008-01711/BMC). LIL is a Pew Latin American Fellow in the Biomedical Sciences supported by The Pew Charitable Trusts. The opinions expressed are those of the authors and do not necessarily reflect the views of The Pew Charitable Trusts.