Publications by authors named "Keith A Stubbs"

99 Publications

Herbicidal activity of fluoroquinolone derivatives.

Plant Direct 2021 Sep 14;5(9):e348. Epub 2021 Sep 14.

School of Molecular Sciences The University of Western Australia Crawley WA Australia.

Development of herbicides with novel modes of action is crucial for weed control and to hinder herbicide resistance. An attractive novel herbicidal target is plant DNA gyrase, which has been demonstrated to be effectively inhibited by the known antimicrobial ciprofloxacin. Despite this good herbicidal activity, ciprofloxacin is not suitable as a herbicide due to its antimicrobial activity; therefore, a diverse library of analogues was analyzed to gain insight into the aspects required for herbicidal activity. This analysis revealed that significant structural modifications were tolerated and that the fluoride at C-6 and a cyclic amino group at C-7 were not crucial for herbicidal activity. The analysis also revealed that these modifications also affected the antibacterial activity with one compound demonstrating good herbicidal activity and weak antibacterial activity, against both Gram-positive and Gram-negative bacteria.
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http://dx.doi.org/10.1002/pld3.348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8438536PMC
September 2021

Antibiotic resistance lessons for the herbicide resistance crisis.

Pest Manag Sci 2021 Sep 18;77(9):3807-3814. Epub 2021 Mar 18.

School of Molecular Sciences, The University of Western Australia, Perth, Australia.

The challenges of resistance to antibiotics and resistance to herbicides have much in common. Antibiotic resistance became a risk in the 1950s, but a concerted global effort to manage it did not begin until after 2000. Widespread herbicide use began during the 1950s and was soon followed by an unabated rise in resistance. Here, we examine what lessons for combatting herbicide resistance could be learnt from the global, coordinated efforts of all stakeholders to avert the antibiotic resistance crisis. © 2021 Society of Chemical Industry.
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http://dx.doi.org/10.1002/ps.6357DOI Listing
September 2021

Anti-Virulence Therapeutic Approaches for .

Antibiotics (Basel) 2021 Jan 21;10(2). Epub 2021 Jan 21.

Marshall Centre for Infectious Disease Research and Training, School of Biomedical Sciences, University of Western Australia, Crawley, WA 6009, Australia.

While antimicrobial resistance (AMR) is seen in both and , the former has become resistant to commonly available over-the-counter antibiotic treatments. It is imperative then to develop new therapies that combat current AMR isolates whilst also circumventing the pathways leading to the development of AMR. This review highlights the growing research interest in developing anti-virulence therapies (AVTs) which are directed towards inhibiting virulence factors to prevent infection. By targeting virulence factors that are not essential for gonococcal survival, it is hypothesized that this will impart a smaller selective pressure for the emergence of resistance in the pathogen and in the microbiome, thus avoiding AMR development to the anti-infective. This review summates the current basis of numerous anti-virulence strategies being explored for .
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http://dx.doi.org/10.3390/antibiotics10020103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7911339PMC
January 2021

Red Algal Molecules - Synthesis of Methyl Neo-β-carrabioside and Its -Linked Variant via Two Synthetic Routes: A Late Stage Ring Closure and Using a 3,6-Anhydro-d-galactosyl Donor.

J Org Chem 2020 12 12;85(24):16182-16195. Epub 2020 Nov 12.

School of Molecular Sciences, The University of Western Australia, Crawley, WA 6009, Australia.

Methyl neo-β-carrabioside has been synthesized for the first time, employing either a late stage ring closure to install the required 3,6-anhydro-bridge or a suitable 3,6-anhydro-galactosyl donor to form the unfavored 1,2--equatorial α-linkage. Using the late stage ring closure approach, an -linked analogue of methyl neo-β-carrabioside was also realized. These compounds have applications in the identification and characterization of marine bacterial -α-3,6-anhydro-d-galactosidases that have specific activity on red algal neo-carrageenan oligosaccharides, such as those found in both family 127 and 129 of the glycoside hydrolases. In addition a biochemical assay using the synthesized methyl neo-β-carrabioside and the marine bacterial -α-3,6-anhydro-d-galactosidase GH129 demonstrates that the minimum substrate unit for the enzyme is neo-β-carrabiose.
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http://dx.doi.org/10.1021/acs.joc.0c02339DOI Listing
December 2020

Vortex Fluidic-Mediated Fabrication of Fast Gelated Silica Hydrogels with Embedded Laccase Nanoflowers for Real-Time Biosensing under Flow.

ACS Appl Mater Interfaces 2020 Nov 5;12(46):51999-52007. Epub 2020 Nov 5.

Flinders Institute for Nanoscale Science and Technology, College of Science and Engineering, Flinders University, Adelaide, South Australia 5042, Australia.

The fabrication of hybrid protein-Cu(PO) nanoflowers (NFs) via an intermediate toroidal structure is dramatically accelerated under shear using a vortex fluidic device (VFD), which possesses a rapidly rotating angled tube. As-prepared laccase NFs (LNFs) exhibit ≈1.8-fold increase in catalytic activity compared to free laccase under diffusion control, which is further enhanced by ≈ 2.9-fold for the catalysis under shear in the VFD. A new LNF immobilization platform, [email protected] incorporated in a VFD tube, was subsequently developed by mixing the LNFs for 15 min with silica hydrogel resulting in gelation along the VFD tube surface. The resulting [email protected] coating is highly stable and reusable, which allows a dramatic 16-fold enhancement in catalytic rates relative to [email protected] inside glass vials. Ultraviolet-visible spectroscopy-based real-time monitoring within the [email protected] tube reveals good stability of the coating in continuous flow processing. The results demonstrate the utility of the VFD microfluidic platform, further highlighting its ability to control chemical and enzymatic processes.
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http://dx.doi.org/10.1021/acsami.0c15669DOI Listing
November 2020

Structure of a GH51 α-L-arabinofuranosidase from Meripilus giganteus: conserved substrate recognition from bacteria to fungi.

Acta Crystallogr D Struct Biol 2020 Nov 16;76(Pt 11):1124-1133. Epub 2020 Oct 16.

York Structural Biology Laboratory, University of York, Heslington, York YO10 5DD, United Kingdom.

α-L-Arabinofuranosidases from glycoside hydrolase family 51 use a stereochemically retaining hydrolytic mechanism to liberate nonreducing terminal α-L-arabinofuranose residues from plant polysaccharides such as arabinoxylan and arabinan. To date, more than ten fungal GH51 α-L-arabinofuranosidases have been functionally characterized, yet no structure of a fungal GH51 enzyme has been solved. In contrast, seven bacterial GH51 enzyme structures, with low sequence similarity to the fungal GH51 enzymes, have been determined. Here, the crystallization and structural characterization of MgGH51, an industrially relevant GH51 α-L-arabinofuranosidase cloned from Meripilus giganteus, are reported. Three crystal forms were grown in different crystallization conditions. The unliganded structure was solved using sulfur SAD data collected from a single crystal using the I23 in vacuo diffraction beamline at Diamond Light Source. Crystal soaks with arabinose, 1,4-dideoxy-1,4-imino-L-arabinitol and two cyclophellitol-derived arabinose mimics reveal a conserved catalytic site and conformational itinerary between fungal and bacterial GH51 α-L-arabinofuranosidases.
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http://dx.doi.org/10.1107/S205979832001253XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7604909PMC
November 2020

Primary antibiotic resistance of among a Chinese Tibetan population.

Future Microbiol 2020 09 9;15:1353-1361. Epub 2020 Sep 9.

West China Marshall Research Center for Infectious Diseases, Center of Infectious Diseases, West China Hospital, Sichuan University, Chengdu, 610041, China.

To evaluate the primary antibiotic resistance in strains isolated from a Chinese Tibetan population. Gastric biopsies from 400 treatment-naive Tibetan patients were collected for isolation. Susceptibility to amoxicillin (AML)/clarithromycin (CLR)/levofloxacin (LEV)/metronidazole (MTZ)/tetracycline (TET)/rifampicin (RIF)/furazolidone (FZD) was determined by E-test or a disk diffusion assay. Biopsies from 117 patients were culture positive (29.3%). The primary resistance rates to MTZ, CLR, LEV, RIF, AML, TET and FZD were 90.6, 44.4, 28.2, 69.2, 7.7, 0.8 and 0.8%, respectively. Interestingly, 42.7% of the strains had simultaneous resistance to CLR and MTZ. Among Tibetan strains, primary resistance rates were high for CLR/MTZ/LEV, whereas primary resistance rates to AML/TET/FZD were low. The high resistance to RIF is a concerning finding.
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http://dx.doi.org/10.2217/fmb-2020-0206DOI Listing
September 2020

Characterisation of an exo-(α-1,3)-3,6-anhydro-d-galactosidase produced by the marine bacterium Zobellia galactanivorans Dsij: Insight into enzyme preference for natural carrageenan oligosaccharides and kinetic characterisation on a novel chromogenic substrate.

Int J Biol Macromol 2020 Nov 5;163:1471-1479. Epub 2020 Aug 5.

CNRS, Sorbonne Université, UMR 8227, Integrative Biology of Marine Models, Station Biologique de Roscoff, CS 90074 Roscoff, Bretagne, France. Electronic address:

Flavobacteriia are important degraders in the marine carbon cycle, due to their ability to efficiently degrade complex algal polysaccharides. A novel exo-(α-1,3)-3,6-anhydro-D-galactosidase activity was recently discovered from a marine Flavobacteriia (Zobellia galactanivorans Dsij) on red algal carrageenan oligosaccharides. The enzyme activity is encoded by a gene found in the first described carrageenan-specific polysaccharide utilization locus (CarPUL) that codes for a family 129 glycoside hydrolase (GH129). The GH129 family is a CAZy family that is strictly partitioned into two niche-based clades: clade 1 contains human host bacterial enzymes and clade 2 contains marine bacterial enzymes. Clade 2 includes the GH129 exo-(α-1,3)-3,6-anhydro-D-galactosidase from Z. galactanivorans (ZgGH129). Despite the discovery of the unique activity for ZgGH129, finer details on the natural substrate specificity for this enzyme are lacking. Examination of enzyme activity on natural carrageenan oligomers using mass spectrometry demonstrated that ZgGH129 hydrolyses terminal 3,6-anhydro-D-galactose from unsulfated non-reducing end neo-β-carrabiose motifs. Due to the lack of chromogenic substrates to examine exo-(α-1,3)-3,6-anhydro-D-galactosidase activity, a novel substrate was synthesised to facilitate the first kinetic characterisation of an exo-(α-1,3)-3,6-anhydro-D-galactosidase, allowing determination of pH and temperature optimums and Michaelis-Menten steady state kinetic data.
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http://dx.doi.org/10.1016/j.ijbiomac.2020.07.298DOI Listing
November 2020

NAG-thiazoline is a potent inhibitor of the Vibrio campbellii GH20 β-N-Acetylglucosaminidase.

FEBS J 2020 11 23;287(22):4982-4995. Epub 2020 Mar 23.

School of Biomolecular Science and Engineering (BSE), Vidyasirimedhi Institute of Science and Technology (VISTEC), Rayong, Thailand.

Vibrio spp. play a vital role in the recycling of chitin in oceans, but several Vibrio strains are highly infectious to aquatic animals and humans. These bacteria require chitin for growth; thus, potent inhibitors of chitin-degrading enzymes could serve as candidate drugs against Vibrio infections. This study examined NAG-thiazoline (NGT)-mediated inhibition of a recombinantly expressed GH20 β-N-acetylglucosaminidase, namely VhGlcNAcase from Vibrio campbellii (formerly V. harveyi) ATCC BAA-1116. NGT strongly inhibited VhGlcNAcase with an IC of 11.9 ± 1.0 μm and K 62 ± 3 µm, respectively. NGT was also found to completely inhibit the growth of V. campbellii strain 650 with an minimal inhibitory concentration value of 0.5 µm. ITC data analysis showed direct binding of NGT to VhGlcNAcase with a K of 32 ± 1.2 μm. The observed ΔG° of -7.56 kcal·mol is the result of a large negative enthalpy change and a small positive entropic compensation, suggesting that NGT binding is enthalpy-driven. The structural complex shows that NGT fully occupies the substrate-binding pocket of VhGlcNAcase and makes an exclusive hydrogen bond network, as well as hydrophobic interactions with the conserved residues around the -1 subsite. Our results strongly suggest that NGT could serve as an excellent scaffold for further development of antimicrobial agents against Vibrio infections. DATABASE: Structural data are available in PDB database under the accession number 6K35.
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http://dx.doi.org/10.1111/febs.15283DOI Listing
November 2020

Genomics-Driven Discovery of Phytotoxic Cytochalasans Involved in the Virulence of the Wheat Pathogen .

ACS Chem Biol 2020 01 20;15(1):226-233. Epub 2019 Dec 20.

Division of Plant Science, Research School of Biology , The Australian National University , Canberra , Australian Capital Territory 2601 , Australia.

The etiology of fungal pathogenesis of grains is critical to global food security. The large number of orphan biosynthetic gene clusters uncovered in fungal plant pathogen genome sequencing projects suggests that we have a significant knowledge gap about the secondary metabolite repertoires of these pathogens and their roles in plant pathogenesis. Cytochalasans are a family of natural products of significant interest due to their ability to bind to actin and interfere with cellular processes that involved actin polymerization; however, our understanding of their biosynthesis and biological roles remains incomplete. Here, we identified a putative polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) gene cluster () that was upregulated in the pathogen during its infection on wheat. Overexpression of the transcription factor gene encoded in the gene cluster resulted in the production of two leucine-derived cytochalasans, phomacins D and E ( and , respectively), and an acetonyl adduct phomacin F. Heterologous expression of the PKS-NRPS gene and the -enoyl reductase (ER) gene in resulted in the production of a novel 2-pyrrolidone precursor prephomacin. Reverse genetics and wheat seedling infection assays showed that Δ mutants exhibited significantly reduced virulence compared to the wild type. We further demonstrated that both and showed potent actin polymerization-inhibitory activities and exhibited potentially monocot-specific antigerminative activities. The findings from this study have advanced our knowledge based on the biosynthesis and biological roles of cytochalasans, the latter of which could have significant implications for our understanding of the molecular mechanisms of fungus-plant interactions.
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http://dx.doi.org/10.1021/acschembio.9b00791DOI Listing
January 2020

East-Asian Helicobacter pylori strains synthesize heptan-deficient lipopolysaccharide.

PLoS Genet 2019 11 20;15(11):e1008497. Epub 2019 Nov 20.

West China Marshall Research Center for Infectious Diseases, Center of Infectious Diseases, Division of Infectious Diseases, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China.

The lipopolysaccharide O-antigen structure expressed by the European Helicobacter pylori model strain G27 encompasses a trisaccharide, an intervening glucan-heptan and distal Lewis antigens that promote immune escape. However, several gaps still remain in the corresponding biosynthetic pathway. Here, systematic mutagenesis of glycosyltransferase genes in G27 combined with lipopolysaccharide structural analysis, uncovered HP0102 as the trisaccharide fucosyltransferase, HP1283 as the heptan transferase, and HP1578 as the GlcNAc transferase that initiates the synthesis of Lewis antigens onto the heptan motif. Comparative genomic analysis of G27 lipopolysaccharide biosynthetic genes in strains of different ethnic origin revealed that East-Asian strains lack the HP1283/HP1578 genes but contain an additional copy of HP1105 and JHP0562. Further correlation of different lipopolysaccharide structures with corresponding gene contents led us to propose that the second copy of HP1105 and the JHP0562 may function as the GlcNAc and Gal transferase, respectively, to initiate synthesis of the Lewis antigen onto the Glc-Trio-Core in East-Asian strains lacking the HP1283/HP1578 genes. In view of the high gastric cancer rate in East Asia, the absence of the HP1283/HP1578 genes in East-Asian H. pylori strains warrants future studies addressing the role of the lipopolysaccharide heptan in pathogenesis.
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http://dx.doi.org/10.1371/journal.pgen.1008497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6892558PMC
November 2019

Biosynthesis of a Tricyclo[6.2.2.0 ]dodecane System by a Berberine Bridge Enzyme-Like Aldolase.

Chemistry 2019 Nov 28;25(66):15062-15066. Epub 2019 Oct 28.

School of Molecular Sciences, The University of Western Australia, Perth, WA, 6009, Australia.

The aldol reaction is one of the most fundamental stereocontrolled carbon-carbon bond-forming reactions and is mainly catalyzed by aldolases in nature. Despite the fact that the aldol reaction has been widely proposed to be involved in fungal secondary metabolite biosynthesis, a dedicated aldolase that catalyzes stereoselective aldol reactions has only rarely been reported in fungi. Herein, we activated a cryptic polyketide biosynthetic gene cluster that was upregulated in the fungal wheat pathogen Parastagonospora nodorum during plant infection; this resulted in the production of the phytotoxic stemphyloxin II (1). Through heterologous reconstruction of the biosynthetic pathway and in vitro assay by using cell-free lysate from Aspergillus nidulans, we demonstrated that a berberine bridge enzyme (BBE)-like protein SthB catalyzes an intramolecular aldol reaction to establish the bridged tricyclo[6.2.2.0 ]dodecane skeleton in the post-assembly tailoring step. The characterization of SthB as an aldolase enriches the catalytic toolbox of classic reactions and the functional diversities of the BBE superfamily of enzymes.
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http://dx.doi.org/10.1002/chem.201904360DOI Listing
November 2019

Heterologous biosynthesis of elsinochrome A sheds light on the formation of the photosensitive perylenequinone system.

Chem Sci 2019 Feb 22;10(5):1457-1465. Epub 2018 Nov 22.

School of Molecular Sciences , University of Western Australia , Perth , WA 6009 , Australia . Email:

Perylenequinones are a class of aromatic polyketides characterised by a highly conjugated pentacyclic core, which confers them with potent light-induced bioactivities and unique photophysical properties. Despite the biosynthetic gene clusters for the perylenequinones elsinochrome A (), cercosporin () and hypocrellin A () being recently identified, key biosynthetic aspects remain elusive. Here, we first expressed the intact gene cluster encoding from the wheat pathogen heterologously in on a yeast-fungal artificial chromosome (YFAC). This led to the identification of a novel flavin-dependent monooxygenase, ElcH, responsible for oxidative enolate coupling of a perylenequinone intermediate to the hexacyclic dihydrobenzo()perylenequinone in . In the absence of ElcH, the perylenequione intermediate formed a hexacyclic cyclohepta()perylenequinone system an intramolecular aldol reaction resulting in and a novel hypocrellin with opposite helicity to . Theoretical calculations supported that and resulted from atropisomerisation upon formation of the 7-membered ring. Using a bottom-up pathway reconstruction approach on a tripartite YFAC system developed in this study, we uncovered that both a berberine bridge enzyme-like oxidase ElcE and a laccase-like multicopper oxidase ElcG are involved in the double coupling of two naphthol intermediates to form the perylenequinone core. Gene swapping with the homologs from the biosynthetic pathway of showed that cognate pairing of the two classes of oxidases is required for the formation of the perylenequinone core, suggesting the involvement of protein-protein interactions.
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http://dx.doi.org/10.1039/c8sc02870bDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6354827PMC
February 2019

Synthetic and Crystallographic Insight into Exploiting sp Hybridization in the Development of α-l-Fucosidase Inhibitors.

Chembiochem 2019 06 18;20(11):1365-1368. Epub 2019 Mar 18.

School of Molecular Sciences, University of Western Australia, 35 Stirling Highway, Crawley, WA, 6009, Australia.

The sugar fucose plays a myriad of roles in biological recognition. Enzymes hydrolyzing fucose from glycoconjugates, α-l-fucosidases, are important targets for inhibitor and probe development. Here we describe the synthesis and evaluation of novel α-l-fucosidase inhibitors, with X-ray crystallographic analysis using an α-l-fucosidase from Bacteroides thetaiotamicron helping to lay a foundation for future development of inhibitors for this important enzyme class.
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http://dx.doi.org/10.1002/cbic.201800710DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6589914PMC
June 2019

Integrating thin film microfluidics in developing a concise synthesis of DGJNAc: A potent inhibitor of α-N-acetylgalctosaminidases.

Bioorg Med Chem Lett 2018 12 11;28(23-24):3748-3751. Epub 2018 Oct 11.

School of Molecular Sciences, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia. Electronic address:

A simple synthesis, which utilizes a thin film microfluidic reactor for a problematic step, of a potent inhibitor of α-N-acetylhexosaminidases, DGJNAc, has been developed.
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http://dx.doi.org/10.1016/j.bmcl.2018.10.015DOI Listing
December 2018

Chemical Ecogenomics-Guided Discovery of Phytotoxic α-Pyrones from the Fungal Wheat Pathogen Parastagonospora nodorum.

Org Lett 2018 10 18;20(19):6148-6152. Epub 2018 Sep 18.

School of Molecular Sciences , The University of Western Australia , Perth , WA 6009 , Australia.

A biosynthetic gene cluster that is significantly upregulated in the fungal wheat pathogen Parastagonospora nodorum during plant infection was reconstructed heterologously in Aspergillus nidulans. This led to the discovery of five new α-pyrone polyketides, alternapyrones B-F (2-6). Compounds 5 and 6, which contain a highly substituted dihydrofuran, exhibited phytotoxicity on wheat seed germination. It is demonstrated that only three enzymes, one highly reducing polyketide synthase and two multifunctional P450 oxygenases, are needed to synthesize the structurally complex products.
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http://dx.doi.org/10.1021/acs.orglett.8b02617DOI Listing
October 2018

Enzyme targets for drug design of new anti-virulence therapeutics.

Curr Opin Struct Biol 2018 12 14;53:140-150. Epub 2018 Sep 14.

School of Molecular Sciences, University of Western Australia, 35 Stirling Highway, Crawley, WA, 6009, Australia; Marshall Centre for Infectious Disease Research and Training, School of Biomedical Sciences, University of Western Australia, 35 Stirling Highway, Crawley, WA, 6009, Australia. Electronic address:

Society has benefitted greatly from the use of antibiotics. Unfortunately, the misuse of these valuable molecules has resulted in increased levels of antibiotic resistance, a major global and public health issue. This resistance and the reliance on a small number of biological targets for the development of antibiotics emphasizes the need for new targets. A critical aspect guiding the development of new antimicrobials through a rational structure-guided approach is to understand the molecular structures of specific biological targets of interest. Here we give an overview of the structures of bacterial virulence enzyme targets involved in protein folding, peptidoglycan biosynthesis and cell wall modification. These include enzymes of the thiol-disulphide oxidoreductase pathway (DSB enzymes), peptidyl-proly cis/trans isomerases (Mips), enzymes from the Mur pathway and enzymes involved in lipopolysaccharide modification (EptA and ArnT). We also present progress towards inhibitor design of these targets for the development of novel anti-virulence therapeutic agents.
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http://dx.doi.org/10.1016/j.sbi.2018.08.010DOI Listing
December 2018

Lipopolysaccharide Structural Differences between Western and Asian Strains.

Toxins (Basel) 2018 09 8;10(9). Epub 2018 Sep 8.

Helicobacter pylori Research Laboratory, School of Biomedical Sciences, Marshall Centre for Infectious Disease Research and Training, University of Western Australia, Nedlands, WA 6009, Australia.

Recent structural analysis of the lipopolysaccharide (LPS) isolated from G27 wild-type and -antigen ligase mutant resulted in the redefinition of the core-oligosaccharide and -antigen domains. The short core-oligosaccharide (Glc⁻Gal⁻Hep-III⁻Hep-II⁻Hep-I⁻KDO) and its attached trisaccharide (Trio, GlcNAc⁻Fuc⁻Hep) appear to be highly conserved structures among strains. The G27 LPS contains a linear glucan⁻heptan linker between the core-Trio and distal Lewis antigens. This linker domain was commonly identified in Western strains. In contrast, out of 12 partial LPS structures of Asian strains, none displayed the heptan moiety, despite the presence of Lewis antigens. This raises the question of how Lewis antigens are attached to the Trio, and whether the LPS structure of Asian strains contain another linker. Of note, a riban was identified as a linker in LPS of the mouse-adapted SS1 strain, suggesting that alternative linker structures can occur. In summary, additional full structural analyses of LPS in Asian strains are required to assess the presence or absence of an alternative linker in these strains. It will also be interesting to study the glucan-heptan linker moieties in pathogenesis as infections in Asia are usually more symptomatic than the ones presented in the Western world.
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http://dx.doi.org/10.3390/toxins10090364DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6162551PMC
September 2018

A mechanism-based GlcNAc-inspired cyclophellitol inactivator of the peptidoglycan recycling enzyme NagZ reverses resistance to β-lactams in Pseudomonas aeruginosa.

Chem Commun (Camb) 2018 Sep 4;54(75):10630-10633. Epub 2018 Sep 4.

School of Molecular Sciences, University of Western Australia, Crawley, WA 6009, Australia.

The development of a potent mechanism-based inactivator of NagZ, an enzyme critical to the production of inducible AmpC β-lactamase in Gram-negative bacteria, is presented. This inactivator significantly reduces MIC values for important β-lactams against a clinically relevant strain of Pseudomonas aeruginosa.
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http://dx.doi.org/10.1039/c8cc05281fDOI Listing
September 2018

Structure of a Talaromyces pinophilus GH62 arabinofuranosidase in complex with AraDNJ at 1.25 Å resolution.

Acta Crystallogr F Struct Biol Commun 2018 Aug 26;74(Pt 8):490-495. Epub 2018 Jul 26.

York Structural Biology Laboratory, Department of Chemistry, The University of York, York YO10 5DD, England.

The enzymatic hydrolysis of complex plant biomass is a major societal goal of the 21st century in order to deliver renewable energy from nonpetroleum and nonfood sources. One of the major problems in many industrial processes, including the production of second-generation biofuels from lignocellulose, is the presence of `hemicelluloses' such as xylans which block access to the cellulosic biomass. Xylans, with a polymeric β-1,4-xylose backbone, are frequently decorated with acetyl, glucuronyl and arabinofuranosyl `side-chain' substituents, all of which need to be removed for complete degradation of the xylan. As such, there is interest in side-chain-cleaving enzymes and their action on polymeric substrates. Here, the 1.25 Å resolution structure of the Talaromyces pinophilus arabinofuranosidase in complex with the inhibitor AraDNJ, which binds with a K of 24 ± 0.4 µM, is reported. Positively charged iminosugars are generally considered to be potent inhibitors of retaining glycosidases by virtue of their ability to interact with both acid/base and nucleophilic carboxylates. Here, AraDNJ shows good inhibition of an inverting enzyme, allowing further insight into the structural basis for arabinoxylan recognition and degradation.
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http://dx.doi.org/10.1107/S2053230X18000250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6096477PMC
August 2018

Targeting plant DIHYDROFOLATE REDUCTASE with antifolates and mechanisms for genetic resistance.

Plant J 2018 Jun 7. Epub 2018 Jun 7.

School of Molecular Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, Perth, 6009, Australia.

The folate biosynthetic pathway and its key enzyme dihydrofolate reductase (DHFR) is a popular target for drug development due to its essential role in the synthesis of DNA precursors and some amino acids. Despite its importance, little is known about plant DHFRs, which, like the enzymes from the malarial parasite Plasmodium, are bifunctional, possessing DHFR and thymidylate synthase (TS) domains. Here using genetic knockout lines we confirmed that either DHFR-TS1 or DHFR-TS2 (but not DHFR-TS3) was essential for seed development. Screening mutated Arabidopsis thaliana seeds for resistance to antimalarial DHFR-inhibitor drugs pyrimethamine and cycloguanil identified causal lesions in DHFR-TS1 and DHFR-TS2, respectively, near the predicted substrate-binding site. The different drug resistance profiles for the plants, enabled by the G137D mutation in DHFR-TS1 and the A71V mutation in DHFR-TS2, were consistent with biochemical studies using recombinant proteins and could be explained by structural models. These findings provide a great improvement in our understanding of plant DHFR-TS and suggest how plant-specific inhibitors might be developed, as DHFR is not currently targeted by commercial herbicides.
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http://dx.doi.org/10.1111/tpj.13983DOI Listing
June 2018

Potent inhibition of a GH20 exo-β-N-acetylglucosaminidase from marine Vibrio bacteria by reaction intermediate analogues.

Int J Biol Macromol 2018 Aug 3;115:1165-1173. Epub 2018 May 3.

Biochemistry-Electrochemistry Research Unit and School of Chemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; Center of Excellence in Advanced Functional Materials, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand. Electronic address:

Exo-β-N-acetylglucosaminidases (GlcNAcases) are hydrolytic enzymes involved in the metabolism of chitin in bacteria and in eukaryotic glycosphingolipid metabolism, with genetic defects in human GlcNAcases (HexA and HexB) resulting in Tay-Sachs and Sandhoff diseases, respectively. Here, we determined the effects of three known inhibitors of exo-β-N-acetylglucosaminidases (PUGNAc, NHAcCAS and NHAcDNJ) on a GH20 exo-β-N-GlcNAcase (VhGlcNAcase) from the pathogenic bacterium Vibrio harveyi, in dose-response experiments. The inhibitors were shown to modify the kinetic parameters (both K and k), yielding significant decreases in the overall efficiency of the enzyme in hydrolyzing the natural substrate diNAG. Molecular interactions between the inhibitors and the enzyme were investigated by isothermal calorimetry (ITC), and were confirmed using molecular docking. VhGlcNAcase was strongly inhibited by these compounds, with PUGNAc having the lowest IC value, of 1.2 μM. Molecular docking suggested that the inhibitors mimicked reaction intermediates, with enzyme-inhibitor interactions being similar to those of the enzyme with diNAG. The equilibrium dissociation constants (K) obtained from ITC were 0.19 μM for PUGNAc, 12.9 μM for NHAcCAS and 25.6 μM for NHAcDNJ, confirming that PUGNAc was the most potent inhibitor. The ITC data indicated that the binding of the enzyme to the inhibitors was driven by enthalpy. The negative heat capacity change (ΔC) of -0.34 ± 0.05 kcal·mol·K indicates that hydrophobic interactions make a substantial contribution to the molecular interactions between PUGNAc and the enzyme. Our results suggest that PUGNAc is a highly potent inhibitor, and suggest its usefulness as a scaffold for potential drugs targeting GlcNAcase-related metabolic diseases.
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http://dx.doi.org/10.1016/j.ijbiomac.2018.04.193DOI Listing
August 2018

Angled Vortex Fluidic Mediated Multicomponent Photocatalytic and Transition Metal-Catalyzed Reactions.

Chemistry 2018 Jun 1;24(35):8869-8874. Epub 2018 Jun 1.

School of Molecular Sciences, University of Western Australia, 35 Stirling Highway, Crawley, WA, 6009, Australia.

The dynamic thin film formed in an angled rapidly rotating tube in a vortex fluidic device (VFD) is effective in facilitating multicomponent reactions (MCRs) as photocatalytic or metal-mediated processes. Here, we demonstrate the utility of the VFD by using two known MCRs, an Ugi-type three component reaction and an A -coupling reaction. The Ugi-type reaction can be done in either confined or continuous-flow modes of operation of the microfluidic platform whereas the A -coupling reaction was optimized for the confined mode of operation. The examples tested gave excellent yields with short reaction times.
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http://dx.doi.org/10.1002/chem.201801109DOI Listing
June 2018

Developing ciprofloxacin analogues against plant DNA gyrase: a novel herbicide mode of action.

Chem Commun (Camb) 2018 Feb;54(15):1869-1872

School of Molecular Sciences, University of Western Australia, Crawley, WA 6009, Australia.

Ciprofloxacin has been shown to exhibit potent herbicidal activity through action against plant DNA gyrase, presenting a novel mode of action. Analogues of ciprofloxacin have been prepared with increased herbicidal activity and diminished antibacterial activity, compared to ciprofloxacin, as demonstrated using model systems.
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http://dx.doi.org/10.1039/c7cc09518jDOI Listing
February 2018

A herbicide structure-activity analysis of the antimalarial lead compound MMV007978 against Arabidopsis thaliana.

Pest Manag Sci 2018 Jul 5;74(7):1558-1563. Epub 2018 Mar 5.

School of Molecular Sciences, University of Western Australia, Crawley, WA, Australia.

Background: To fight herbicide-resistant weeds, new herbicides are needed; particularly ones with new modes of action. Building on the revelation that many antimalarial drugs are herbicidal, here we focus on the Medicines for Malaria Venture antimalarial lead compound MMV007978 that has herbicidal activity against the model plant Arabidopsis thaliana.

Results: Twenty-two variations of the lead compound thiophenyl motif revealed that change was tolerated provided ring size and charge were retained. MMV007978 was active against select monocot and dicot weeds, and physiological profiling indicated that its mode of action is related to germination and cell division. Of interest is the fact that the compound has a profile that is currently not found among known herbicides.

Conclusion: We demonstrate that the antimalarial compound MMV007978 is also herbicidal and that exploiting lead compounds that are often understudied could lead to the identification of interesting herbicidal scaffolds. Further structural investigation of MMV007978 could provide improved herbicidal chemistries with a potential new mode of action. © 2018 Society of Chemical Industry.
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http://dx.doi.org/10.1002/ps.4872DOI Listing
July 2018

Carrageenan catabolism is encoded by a complex regulon in marine heterotrophic bacteria.

Nat Commun 2017 11 22;8(1):1685. Epub 2017 Nov 22.

Sorbonne Universités, UPMC Univ Paris 06, CNRS, UMR 8227, Integrative Biology of Marine Models, Station Biologique de Roscoff, CS 90074, Roscoff, Bretagne, France.

Macroalgae contribute substantially to primary production in coastal ecosystems. Their biomass, mainly consisting of polysaccharides, is cycled into the environment by marine heterotrophic bacteria using largely uncharacterized mechanisms. Here we describe the complete catabolic pathway for carrageenans, major cell wall polysaccharides of red macroalgae, in the marine heterotrophic bacterium Zobellia galactanivorans. Carrageenan catabolism relies on a multifaceted carrageenan-induced regulon, including a non-canonical polysaccharide utilization locus (PUL) and genes distal to the PUL, including a susCD-like pair. The carrageenan utilization system is well conserved in marine Bacteroidetes but modified in other phyla of marine heterotrophic bacteria. The core system is completed by additional functions that might be assumed by non-orthologous genes in different species. This complex genetic structure may be the result of multiple evolutionary events including gene duplications and horizontal gene transfers. These results allow for an extension on the definition of bacterial PUL-mediated polysaccharide digestion.
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http://dx.doi.org/10.1038/s41467-017-01832-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5698469PMC
November 2017

Insights from the redefinition of lipopolysaccharide O-antigen and core-oligosaccharide domains.

Microb Cell 2017 Apr 25;4(5):175-178. Epub 2017 Apr 25.

Helicobacter pylori Research Laboratory, School of Pathology & Laboratory Medicine, Marshall Centre for Infectious Disease Research and Training, University of Western Australia, Nedlands, Australia.

is a Gram-negative extracellular bacterium, first discovered by the Australian physicians Barry Marshall and Robin Warren in 1982, that colonises the human stomach mucosa. It is the leading cause of peptic ulcer and commonly infects humans worldwide with prevalence as high as 90% in some countries. infection usually results in asymptomatic chronic gastritis, however 10-15% of cases develop duodenal or gastric ulcers and 1-3% develop stomach cancer. Infection is generally acquired during childhood and persists for life in the absence of antibiotic treatment. has had a long period of co-evolution with humans, going back to human migration out of Africa. This prolonged relationship is likely to have shaped the overall host-pathogen interactions and repertoire of virulence strategies which employs to establish robust colonisation, escape immune responses and persist in the gastric niche. In this regard, lipopolysaccharide (LPS) is a key surface determinant in establishing colonisation and persistence via host mimicry and resistance to cationic antimicrobial peptides. Thus, elucidation of the LPS structure and corresponding biosynthetic pathway represents an important step towards better understanding of pathogenesis and the development of novel therapeutic interventions.
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http://dx.doi.org/10.15698/mic2017.05.574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5425279PMC
April 2017

Exploiting the Evolutionary Relationship between Malarial Parasites and Plants To Develop New Herbicides.

Angew Chem Int Ed Engl 2017 08 12;56(33):9881-9885. Epub 2017 Jul 12.

School of Molecular Sciences, University of Western Australia, 35 Stirling Highway, Crawley, WA, 6009, Australia.

Herbicide resistance is driving a need to develop new herbicides. The evolutionary relationship between apicomplexan parasites, such as those causing malaria, and plants is close enough that many antimalarial drugs are herbicidal and so represent novel scaffolds for herbicide development. Using a compound library from the Medicines for Malaria Venture, the model plant Arabidopsis thaliana, and a physicochemical database of known herbicides, a compound was discovered that showed post-emergence herbicidal activity equal to commercial herbicides. Using structure-activity analysis, important points for its potency were found. The compound was also tested and found to be active against common crop weeds. Physiological profiling suggested the compound was a photosystem II inhibitor, representing a new scaffold for herbicide development. Overall this approach demonstrates the viability of using antimalarial compounds as lead compounds for the development of much needed new herbicides.
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http://dx.doi.org/10.1002/anie.201705400DOI Listing
August 2017

Helicobacter pylori gene silencing in vivo demonstrates urease is essential for chronic infection.

PLoS Pathog 2017 Jun 23;13(6):e1006464. Epub 2017 Jun 23.

Helicobacter pylori Research Laboratory, Marshall Centre for Infectious Disease Research and Training, School of Biomedical Sciences, University of Western Australia, Nedlands, Western Australia, Australia.

Helicobacter pylori infection causes chronic active gastritis that after many years of infection can develop into peptic ulceration or gastric adenocarcinoma. The bacterium is highly adapted to surviving in the gastric environment and a key adaptation is the virulence factor urease. Although widely postulated, the requirement of urease expression for persistent infection has not been elucidated experimentally as conventional urease knockout mutants are incapable of colonization. To overcome this constraint, conditional H. pylori urease mutants were constructed by adapting the tetracycline inducible expression system that enabled changing the urease phenotype of the bacteria during established infection. Through tight regulation we demonstrate that urease expression is not only required for establishing initial colonization but also for maintaining chronic infection. Furthermore, successful isolation of tet-escape mutants from a late infection time point revealed the strong selective pressure on this gastric pathogen to continuously express urease in order to maintain chronic infection. In addition to mutations in the conditional gene expression system, escape mutants were found to harbor changes in other genes including the alternative RNA polymerase sigma factor, fliA, highlighting the genetic plasticity of H. pylori to adapt to a changing niche. The tet-system described here opens up opportunities to studying genes involved in the chronic stage of H. pylori infection to gain insight into bacterial mechanisms promoting immune escape and life-long infection. Furthermore, this genetic tool also allows for a new avenue of inquiry into understanding the importance of various virulence determinants in a changing biological environment when the bacterium is put under duress.
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http://dx.doi.org/10.1371/journal.ppat.1006464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5500380PMC
June 2017

Heterologous expression of cytotoxic sesquiterpenoids from the medicinal mushroom Lignosus rhinocerotis in yeast.

Microb Cell Fact 2017 Jun 12;16(1):103. Epub 2017 Jun 12.

Research School of Biology, The Australian National University, Canberra, Australia.

Background: Genome mining facilitated by heterologous systems is an emerging approach to access the chemical diversity encoded in basidiomycete genomes. In this study, three sesquiterpene synthase genes, GME3634, GME3638, and GME9210, which were highly expressed in the sclerotium of the medicinal mushroom Lignosus rhinocerotis, were cloned and heterologously expressed in a yeast system.

Results: Metabolite profile analysis of the yeast culture extracts by GC-MS showed the production of several sesquiterpene alcohols (CHO), including cadinols and germacrene D-4-ol as major products. Other detected sesquiterpenes include selina-6-en-4-ol, β-elemene, β-cubebene, and cedrene. Two purified major compounds namely (+)-torreyol and α-cadinol synthesised by GME3638 and GME3634 respectively, are stereoisomers and their chemical structures were confirmed by H and C NMR. Phylogenetic analysis revealed that GME3638 and GME3634 are a pair of orthologues, and are grouped together with terpene synthases that synthesise cadinenes and related sesquiterpenes. (+)-Torreyol and α-cadinol were tested against a panel of human cancer cell lines and the latter was found to exhibit selective potent cytotoxicity in breast adenocarcinoma cells (MCF7) with IC value of 3.5 ± 0.58 μg/ml while α-cadinol is less active (IC = 18.0 ± 3.27 μg/ml).

Conclusions: This demonstrates that yeast-based genome mining, guided by transcriptomics, is a promising approach for uncovering bioactive compounds from medicinal mushrooms.
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http://dx.doi.org/10.1186/s12934-017-0713-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5468996PMC
June 2017
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