The Structure of Sedoheptulose-7-Phosphate Isomerase from Burkholderia pseudomallei Reveals a Zinc Binding Site at the Heart of the Active Site

Harmer, Nicholas J.

doi:10.1016/j.jmb.2010.04.058

Cited by 19 publications

(53 citation statements)

References 42 publications

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“…2C). To assess whether this elevated protein content was due to cell lysis, the supernatants were also probed with antibodies against cytoplasmic proteins GmhA, Obg, and Zwf (54)(55)(56)(57). During growth in GCBL medium, the Δngo1985 and ΔbamE GC mutants had elevated amounts of all cytoplasmic protein markers compared to the WT (Fig.…”

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confidence: 99%

Deciphering the Function of New Gonococcal Vaccine Antigens Using Phenotypic Microarrays

et al. 2017

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The function and extracellular location of cell envelope proteins make them attractive candidates for developing vaccines against bacterial diseases, including challenging drug-resistant pathogens, such as Neisseria gonorrhoeae. A proteomicsdriven reverse vaccinology approach has delivered multiple gonorrhea vaccine candidates; however, the biological functions of many of them remain to be elucidated. Herein, the functions of six gonorrhea vaccine candidates-NGO2121, NGO1985, NGO2054, NGO2111, NGO1205, and NGO1344 -in cell envelope homeostasis were probed using phenotype microarrays under 1,056 conditions and a ΔbamE mutant (Δngo1780) as a reference of perturbed outer membrane integrity. Optimal growth conditions for an N. gonorrhoeae phenotype microarray assay in defined liquid medium were developed, which can be useful in other applications, including rapid and thorough antimicrobial susceptibility assessment. Our studies revealed 91 conditions having uniquely positive or negative effects on one of the examined mutants. A cluster analysis of 37 and 57 commonly beneficial and detrimental compounds, respectively, revealed three separate phenotype groups: NGO2121 and NGO1985; NGO1344 and BamE; and the trio of NGO1205, NGO2111, and NGO2054, with the last protein forming an independent branch of this cluster. Similar phenotypes were associated with loss of these vaccine candidates in the highly antibiotic-resistant WHO X strain. Based on their extensive sensitivity phenomes, NGO1985 and NGO2121 appear to be the most promising vaccine candidates. This study establishes the principle that phenotype microarrays can be successfully applied to a fastidious bacterial organism, such as N. gonorrhoeae. IMPORTANCE Innovative approaches are required to develop vaccines against prevalent and neglected sexually transmitted infections, such as gonorrhea. Herein, we have utilized phenotype microarrays in the first such investigation into Neisseria gonorrhoeae to probe the function of proteome-derived vaccine candidates in cell envelope homeostasis. Information gained from this screening can feed the vaccine candidate decision tree by providing insights into the roles these proteins play in membrane permeability, integrity, and overall N. gonorrhoeae physiology. The optimized screening protocol can be applied in investigations into the function of other hypothetical proteins of N. gonorrhoeae discovered in the expanding number of whole-genome sequences, in addition to revealing phenotypic differences between clinical and laboratory strains.KEYWORDS Neisseria gonorrhoeae, vaccine antigens, cell envelope, phenotypic microarrays, antibiotics, Biolog I n Gram-negative bacteria, the cell envelope is composed of the outer membrane, the cell wall, and the cytoplasmic or inner membrane, each of which can be further broken down into its constituents (1). The outer membrane is a bilayer composed of a lipopolysaccharide (LPS) or lipooligosaccharide (LOS) outer leaflet facing the extracel-

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confidence: 99%

Deciphering the Function of New Gonococcal Vaccine Antigens Using Phenotypic Microarrays

et al. 2017

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“…Although a mechanism involving a hydride shift has not been totally ruled out, a double tautomerization involving an intermediate 1,2-enediol seems likely, as proposed by Junop [28] and Harmer [29] (see Scheme 7). Thus, low-micromolar inhibition levels were measured for the gluco analogue 9 of mannoheptose H7P 2.…”

Section: Resultsmentioning

confidence: 99%

“…It was suggested [29] that the closed conformation of B. pseudomalleis GmhA is catalytically relevant for the development of therapeutics. To date eight structures of the isomerase orthologues of 5 bacterial species are reported in the literature.…”

Section: Resultsmentioning

confidence: 99%

“…[25] Later on, some of us identified inhibitors of HldE from a high-throughput screening that allowed a structure activity relationship studies on heterocyclic structures. [28][29][30] However, to the best of our knowledge, there is no GmhA inhibitor reported to date. [28][29][30] However, to the best of our knowledge, there is no GmhA inhibitor reported to date.…”

Section: Introductionmentioning

confidence: 99%

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Systematic Synthesis of Inhibitors of the Two First Enzymes of the Bacterial Heptose Biosynthetic Pathway: Towards Antivirulence Molecules Targeting Lipopolysaccharide Biosynthesis

Durka

Tikad

Périon

et al. 2011

Chemistry A European J

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L-Heptoses (L-glycero-D-manno-heptopyranoses) are constituents of the inner core of lipolysaccharide (LPS), a molecule playing key roles in the mortality of many infectious diseases as well as in the virulence of many human pathogens. The inhibition of the first enzymes of the bacterial heptose biosynthetic pathway is an almost unexplored field to date although it appears to be a very novel way for the development of antivirulence drugs. We report the synthesis of a series of D-glycero-D-manno-heptopyranose 7-phosphate (H7P) analogues and their inhibition properties against the isomerase GmhA and the the kinase HldE, the two first enzymes of the bacterial heptose biosynthetic pathway. The heptose structures have been modified at the 1-, 2-, 6- and 7-positions to probe the importance of the key structural features of H7P that allow a tight binding to the target enzymes; H7P being the product of GmhA and the substrate of HldE, the second objective was to find structures that could simultaneously inhibit both enzymes. We found that GmhA and HldE were extremely sensitive to structural modifications at the 6- and 7- positions of the heptose scaffold. To our surprise, the epimeric analogue of H7P displaying a D-glucopyranose configuration was found to be the best inhibitor of both enzymes but also the only molecule of this series that could inhibit GmhA (IC(50)=34 μM) and HldE (IC(50)=9.4 μM) in the low micromolar range. Noteworthy, this study describes the first inhibitors of GmhA ever reported, and paves the way to the design of a second generation of molecules targeting the bacterial virulence.

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“…[23] Very recently, however, a modified mechanism has been proposed for Burkolderia pseudomallei GmhA: this enzyme contains in the active site a Zn 2 + ion that orients the side chains of Glu68 and Gln175, acting as the base and the acid respectively, to promote the isomerisation reaction. [24] In this work we report the characterisation of the kdsD homologue from P. aeruginosa and provide evidence that the gene is essential for growth. Moreover, on the basis of the recently reported E. coli KdsD structural data, we identify key amino acid residues for the activity of the P. aeruginosa homologue.…”

Section: Introductionmentioning

confidence: 92%

Targeting Bacterial Membranes: Identification of Pseudomonas aeruginosaD‐Arabinose‐5P Isomerase and NMR Characterisation of its Substrate Recognition and Binding Properties

et al. 2011

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The identification and characterisation of Pseudomonas aeruginosa KdsD (Pa-KdsD), a D-arabinose-5P isomerase involved in the biosynthesis of 3-deoxy-D-manno-oct-2-ulosonic acid and thus of lipopolysaccharide (LPS), are reported. We have demonstrated that KdsD is essential for P. aeruginosa survival and thus represents a key target for the development of novel antibacterial drugs. The key amino acid residues for protein activity have been identified. The structural requirements for substrate recognition and binding have been characterised for the wild-type protein, and the effect of mutations of the key residues on catalytic activity and binding have been evaluated by saturation transfer difference (STD) NMR spectroscopy. Our data provide important structural information for the rational design of new KdsD inhibitors as potential antibacterial drugs.

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The Structure of Sedoheptulose-7-Phosphate Isomerase from Burkholderia pseudomallei Reveals a Zinc Binding Site at the Heart of the Active Site

Cited by 19 publications

References 42 publications

Deciphering the Function of New Gonococcal Vaccine Antigens Using Phenotypic Microarrays

Deciphering the Function of New Gonococcal Vaccine Antigens Using Phenotypic Microarrays

Systematic Synthesis of Inhibitors of the Two First Enzymes of the Bacterial Heptose Biosynthetic Pathway: Towards Antivirulence Molecules Targeting Lipopolysaccharide Biosynthesis

Targeting Bacterial Membranes: Identification of Pseudomonas aeruginosaD‐Arabinose‐5P Isomerase and NMR Characterisation of its Substrate Recognition and Binding Properties

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