Bacterial polysaccharide synthesis and export Woodward, Laura; Naismith, James H
Current opinion in structural biology,
October 2016, 2016-Oct, 2016-10-00, 20161001, Letnik:
40
Journal Article
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•In bacteria carbohydrate polymers undergo a journey from inside to outside the cell.•Many membrane proteins are key to the synthesis and translocation of the polymers.•Structural information on the ...process is becoming increasingly rich.
All domains of life make carbohydrate polymers and by anchoring them to lipid molecules they can decorate the outside of the cell. Polysaccharides are linked to proteins by glycosylation, a process found in both bacteria and in higher organisms. Bacteria do have other distinct uses for carbohydrate polymers; in gram-negative bacteria glycolipids form the outer leaflet of the outer membrane and in many pathogens (both gram-positive and gram-negative) sugar polymers are used to build a capsule or are secreted into the environment. There are parallels, but of course differences, in the biosynthesis of glycolipids between prokaryotes and eukaryotes, which occur at the membrane. The translocation of large sugar polymers across the outer membrane is unique to gram-negative bacteria. Recent progress in the molecular understanding of both the biosynthesis at the inner membrane and the translocation across the outer membrane are reviewed here.
The conjugation of siderophores to antimicrobial molecules is an attractive strategy to overcome the low outer membrane permeability of Gram-negative bacteria. In this Trojan horse approach, the ...transport of drug conjugates is redirected via TonB-dependent receptors (TBDR), which are involved in the uptake of essential nutrients, including iron. Previous reports have demonstrated the involvement of the TBDRs PiuA and PirA from
and their orthologues in
in the uptake of siderophore-beta-lactam drug conjugates. By
screening, we further identified a PiuA orthologue, termed PiuD, present in clinical isolates, including strain LESB58. The
gene in LESB58 is located at the same genetic locus as
in strain PAO1. PiuD has a similar crystal structure as PiuA and is involved in the transport of the siderophore-drug conjugates BAL30072, MC-1, and cefiderocol in strain LESB58. To screen for additional siderophore-drug uptake systems, we overexpressed 28 of the 34 TBDRs of strain PAO1 and identified PfuA, OptE, OptJ, and the pyochelin receptor FptA as novel TBDRs conferring increased susceptibility to siderophore-drug conjugates. The existence of a TBDR repertoire in
able to transport siderophore-drug molecules potentially decreases the likelihood of resistance emergence during therapy.
Bacteria use small molecules called siderophores to scavenge iron. Siderophore-Fe
complexes are recognised by outer-membrane transporters and imported into the periplasm in a process dependent on the ...inner-membrane protein TonB. The siderophore enterobactin is secreted by members of the family Enterobacteriaceae, but many other bacteria including Pseudomonas species can use it. Here, we show that the Pseudomonas transporter PfeA recognises enterobactin using extracellular loops distant from the pore. The relevance of this site is supported by in vivo and in vitro analyses. We suggest there is a second binding site deeper inside the structure and propose that correlated changes in hydrogen bonds link binding-induced structural re-arrangements to the structural adjustment of the periplasmic TonB-binding motif.
Diets high in sugar are recognized as a serious health problem, and there is a drive to reduce their consumption. Steviol glycosides are natural zero-calorie sweeteners, but the most desirable ones ...are biosynthesized with low yields. UGT76G1 catalyzes the β (1-3) addition of glucose to steviol glycosides, which gives them the preferred taste. UGT76G1 is able to transfer glucose to multiple steviol substrates yet remains highly specific in the glycosidic linkage it creates. Here, we report multiple complex structures of the enzyme combined with biochemical data, which reveal that the enzyme utilizes hydrophobic interactions for substrate recognition. The lack of a strict three-dimensional recognition arrangement, typical of hydrogen bonds, permits two different orientations for β (1-3) sugar addition. The use of hydrophobic recognition is unusual in a regio- and stereo-specific catalysis. Harnessing such non-specific hydrophobic interactions could have wide applications in the synthesis of complex glycoconjugates.
Gram-negative bacteria and their complex cell envelope, which comprises an outer membrane and an inner membrane, are an important and attractive system for studying the translocation of small ...molecules across biological membranes. In the outer membrane of Enterobacteriaceae, trimeric porins control the cellular uptake of small molecules, including nutrients and antibacterial agents. The relatively slow porin-mediated passive uptake across the outer membrane and active efflux via efflux pumps in the inner membrane creates a permeability barrier. The synergistic action of outer membrane permeability, efflux pump activities and enzymatic degradation efficiently reduces the intracellular concentrations of small molecules and contributes to the emergence of antibiotic resistance. In this Review, we discuss recent advances in our understanding of the molecular and functional roles of general porins in small-molecule translocation in Enterobacteriaceae and consider the crucial contribution of porins in antibiotic resistance.
Regioselective modification of amino acids within the context of a peptide is common to a number of biosynthetic pathways, and many of the resulting products have potential as therapeutics. The ...ATP-dependent enzyme LynD heterocyclizes multiple cysteine residues to thiazolines within a peptide substrate. The enzyme requires the substrate to have a conserved N-terminal leader for full activity. Catalysis is almost insensitive to immediately flanking residues in the substrate, suggesting that recognition occurs distant from the active site. Nucleotide and peptide substrate co-complex structures of LynD reveal that the substrate leader peptide binds to and extends the β-sheet of a conserved domain of LynD, whereas catalysis is accomplished in another conserved domain. The spatial segregation of catalysis from recognition combines seemingly contradictory properties of regioselectivity and promiscuity, and it appears to be a conserved strategy in other peptide-modifying enzymes. A variant of LynD that efficiently processes substrates without a leader peptide has been engineered.
The purpose of this review was to identify the effectiveness of environmental control (EC) non-pharmaceutical interventions (NPIs) in reducing transmission of SARS-CoV-2 through conducting a ...systematic review. EC NPIs considered in this review are room ventilation, air filtration/cleaning, room occupancy, surface disinfection, barrier devices,
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monitoring and one-way-systems. Systematic searches of databases from Web of Science, Medline, EMBASE, preprint servers MedRxiv and BioRxiv were conducted in order to identify studies reported between 1 January 2020 and 1 December 2022. All articles reporting on the effectiveness of ventilation, air filtration/cleaning, room occupancy, surface disinfection, barrier devices,
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monitoring and one-way systems in reducing transmission of SARS-CoV-2 were retrieved and screened. In total, 13 971 articles were identified for screening. The initial title and abstract screening identified 1328 articles for full text review. Overall, 19 references provided evidence for the effectiveness of NPIs: 12 reported on ventilation, 4 on air cleaning devices, 5 on surface disinfection, 6 on room occupancy and 1 on screens/barriers. No studies were found that considered the effectiveness of
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monitoring or the implementation of one-way systems. Many of these studies were assessed to have critical risk of bias in at least one domain, largely due to confounding factors that could have affected the measured outcomes. As a result, there is low confidence in the findings. Evidence suggests that EC NPIs of ventilation, air cleaning devices and reduction in room-occupancy may have a role in reducing transmission in certain settings. However, the evidence was usually of low or very low quality and certainty, and hence the level of confidence ascribed to this conclusion is low. Based on the evidence found, it was not possible to draw any specific conclusions regarding the effectiveness of surface disinfection and the use of barrier devices. From these results, we further conclude that community agreed standards for well-designed epidemiological studies with low risk of bias are needed. Implementation of such standards would enable more confident assessment in the future of the effectiveness of EC NPIs in reducing transmission of SARS-CoV-2 and other pathogens in real-world settings.
This article is part of the theme issue ‘The effectiveness of non-pharmaceutical interventions on the COVID-19 pandemic: the evidence’.
Molecules that alter the normal dynamics of microtubule assembly and disassembly include many anticancer drugs in clinical use. So far all such therapeutics target β-tubulin, and structural biology ...has explained the basis of their action and permitted design of new drugs. However, by shifting the profile of β-tubulin isoforms, cancer cells become resistant to treatment. Compounds that bind to α-tubulin are less well characterized and unexploited. The natural product pironetin is known to bind to α-tubulin and is a potent inhibitor of microtubule polymerization. Previous reports had identified that pironetin reacts with lysine-352 residue however analogues designed on this model had much lower potency, which was difficult to explain, hindering further development. We report crystallographic and mass spectrometric data that reveal that pironetin forms a covalent bond to cysteine-316 in α-tubulin via a Michael addition reaction. These data provide a basis for the rational design of α-tubulin targeting chemotherapeutics.
The macrocyclization of linear peptides is very often accompanied by significant improvements in their stability and biological activity. Many strategies are available for their chemical ...macrocyclization, however, enzyme‐mediated methods remain of great interest in terms of synthetic utility. To date, known macrocyclization enzymes have been shown to be active on both peptide and protein substrates. Here we show that the macrocyclization enzyme of the cyanobactin family, PatGmac, is capable of macrocyclizing substrates with one, two, or three 1,4‐substituted 1,2,3‐triazole moieties. The introduction of non‐peptidic scaffolds into macrocycles is highly desirable in tuning the activity and physical properties of peptidic macrocycles. We have isolated and fully characterized nine non‐natural triazole‐containing cyclic peptides, a further ten molecules are also synthesized. PatGmac has now been shown to be an effective and versatile tool for the ring closure by peptide bond formation.
The macrocyclase enzyme PatGmac from the patellamide pathway of the cyanobactin family successfully macrocyclized non‐natural peptides where one, two, or three 1,4‐substituted 1,2,3‐triazole rings were incorporated at different positions of the core peptide. 19 cyclic peptides were macrocyclized by PatGmac, among which 9 were isolated and fully characterized.
Structural biology studies inside cells and tissues require methods to thin vitrified specimens to electron transparency. Until now, focused ion beams based on gallium have been used. However, ion ...implantation, changes to surface chemistry and an inability to access high currents limit gallium application. Here, we show that plasma-coupled ion sources can produce cryogenic lamellae of vitrified human cells in a robust and automated manner, with quality sufficient for pseudo-atomic structure determination. Lamellae were produced in a prototype microscope equipped for long cryogenic run times (> 1 week) and with multi-specimen support fully compatible with modern-day transmission electron microscopes. We demonstrate that plasma ion sources can be used for structural biology within cells, determining a structure in situ to 4.9 Å, and characterise the resolution dependence on particle distance from the lamella edge. We describe a workflow upon which different plasmas can be examined to further streamline lamella fabrication.
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