Structural Studies of FlaA1 from Helicobacter pylori Reveal the Mechanism for Inverting 4,6-Dehydratase Activity

Ishiyama, Noboru; Creuzenet, Carole; Miller, Wayne L.; Demendi, Melinda; Anderson, Erin M.; Harauz, George; Lam, Joseph S.; Berghuis, Albert M.

doi:10.1074/jbc.m602393200

Cited by 49 publications

(80 citation statements)

References 49 publications

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“…Thus, Z= is reflective of both the assay signal dynamic range and the data variation associated with the signal measurements and is used for comparison and evaluation of the quality of assays. A high-throughput assay with a 1 Ͼ Z= Ͼ 0.5 score is considered an excellent assay (28). Therefore, results from the plates with Z= Ͼ0.5 were analyzed.…”

Section: Resultsmentioning

confidence: 99%

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Small-Molecule Inhibitors of the Pseudaminic Acid Biosynthetic Pathway: Targeting Motility as a Key Bacterial Virulence Factor

Ménard

Schoenhofen

Lin

et al. 2014

Antimicrob Agents Chemother

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Helicobacter pylori is motile by means of polar flagella, and this motility has been shown to play a critical role in pathogenicity. The major structural flagellin proteins have been shown to be glycosylated with the nonulosonate sugar, pseudaminic acid (Pse). This glycan is unique to microorganisms, and the process of flagellin glycosylation is required for H. pylori flagellar assembly and consequent motility. As such, the Pse biosynthetic pathway offers considerable potential as an antivirulence drug target, especially since motility is required for H. pylori colonization and persistence in the host. This report describes screening the five Pse biosynthetic enzymes for small-molecule inhibitors using both high-throughput screening (HTS) and in silico (virtual screening [VS]) approaches. Using a 100,000-compound library, 1,773 hits that exhibited a 40% threshold inhibition at a 10 M concentration were identified by HTS. In addition, VS efforts using a 1.6-million compound library directed at two pathway enzymes identified 80 hits, 4 of which exhibited reasonable inhibition at a 10 M concentration in vitro. Further secondary screening which identified 320 unique molecular structures or validated hits was performed. Following kinetic studies and structureactivity relationship (SAR) analysis of selected inhibitors from our refined list of 320 compounds, we demonstrated that three inhibitors with 50% inhibitory concentrations (IC 50 s) of approximately 14 M, which belonged to a distinct chemical cluster, were able to penetrate the Gram-negative cell membrane and prevent formation of flagella.

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Section: Resultsmentioning

confidence: 99%

“…In addition, it has been demonstrated that all five Pse pathway enzymes can be combined in a single one-pot reaction for the synthesis of Pse using UDPGlcNAc as an initial substrate (22). Structural studies of three of the biosynthetic enzymes have also been completed (26)(27)(28).…”

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confidence: 99%

Small-Molecule Inhibitors of the Pseudaminic Acid Biosynthetic Pathway: Targeting Motility as a Key Bacterial Virulence Factor

Ménard

Schoenhofen

Lin

et al. 2014

Antimicrob Agents Chemother

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“…Two homologous proteins, FlaA1 and PseB (46% and 49% similarity to the B. subtilis NDmed YpqP protein, respectively) (see Fig. S1 in the supplemental material), have been characterized biochemically as UDP-N-acetylglucosamine 5-inverting 4,6-dehydratases, which catalyze the first step in the biosynthesis of pseudaminic acid in Helicobacter pylori and Campylobacter jejuni (60,61). Complementary analytical analysis would be needed to determine if B. subtilis can produce pseudaminic acid.…”

Section: Discussionmentioning

confidence: 99%

Identification of ypqP as a New Bacillus subtilis Biofilm Determinant That Mediates the Protection of Staphylococcus aureus against Antimicrobial Agents in Mixed-Species Communities

Sanchez-Vizuete

Coq

Bridier³

et al. 2015

Appl Environ Microbiol

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In most habitats, microbial life is organized in biofilms, three-dimensional edifices sustained by extracellular polymeric substances that enable bacteria to resist harsh and changing environments. Under multispecies conditions, bacteria can benefit from the polymers produced by other species ("public goods"), thus improving their survival under toxic conditions. A recent study showed that a Bacillus subtilis hospital isolate (NDmed) was able to protect Staphylococcus aureus from biocide action in multispecies biofilms. In this work, we identified ypqP, a gene whose product is required in NDmed for thick-biofilm formation on submerged surfaces and for resistance to two biocides widely used in hospitals. NDmed and S. aureus formed mixed biofilms, and both their spatial arrangement and pathogen protection were mediated by YpqP. Functional ypqP is present in other natural B. subtilis biofilm-forming isolates. However, the gene is disrupted by the SP␤ prophage in the weak submerged-biofilm-forming strains NCIB3610 and 168, which are both less resistant than NDmed to the biocides tested. Furthermore, in a 168 laboratory strain cured of the SP␤ prophage, the reestablishment of a functional ypqP gene led to increased thickness and resistance to biocides of the associated biofilms. We therefore propose that YpqP is a new and important determinant of B. subtilis surface biofilm architecture, protection against exposure to toxic compounds, and social behavior in bacterial communities. Bacillus subtilis is a nonpathogenic Gram-positive bacterium that can be found in its natural habitats as free cells or associated with surfaces in biofilms. In the soil, B. subtilis strains have been shown to form surface-associated communities on plant tissues that protect them from infection by pathogens (1-3). Because of its ability to resist stress through biofilm or spore formation, the bacterium has been isolated from extreme environments, such as sand deserts, clouds, and the digestive tracts of animals (4 -6). As well as its ubiquitous presence in diverse habitats, B. subtilis has been used extensively in biotechnological applications, such as the production of natto, a traditional Japanese food made of fermented soybeans (7), and the production of industrial enzymes and pharmaceutical proteins (8, 9). As a generally recognized as safe (GRAS) organism, B. subtilis is also used as a biocontrol agent in agriculture and livestock buildings (10 -14) and as a probiotic agent to improve human and animal health by preventing gastrointestinal infections (15,16).In fundamental research, B. subtilis has emerged as the model organism for deciphering the complex genetic regulation involved in the biofilm mode of life of Gram-positive bacteria. The domesticated strain B. subtilis 168 has been widely used to dissect metabolic and cellular processes. However, this strain is not able to form robust pellicles and complex colonies like those of its parental strain, NCIB3610, a descendant of the original Marburg strain that was deposited in 1951 (17)....

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“…Recent data indicate that, in addition to UDP-4-keto-6-deoxy-GlcNAc, Cj1293 also generates UDP-2-acetamido-2,6-dideoxy-␤-L-arabino-4-hexulose (25) via a change of chirality at carbon 5 during the dehydration step. Structural studies of a homologous enzyme from Helicobacter pylori, FlaA1, indicate that the formation of the 4-keto-arabino intermediate is enzymatically catalyzed but that of the 4-keto-gluco intermediate is not and occurs via enolization of a double bond between C-4 and C-5 (26) (Fig. 1).…”

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confidence: 99%

Cj1121c, a Novel UDP-4-keto-6-deoxy-GlcNAc C-4 Aminotransferase Essential for Protein Glycosylation and Virulence in Campylobacter jejuni

Somalinga

Merkx-Jacques

Ratnayake

et al. 2006

Journal of Biological Chemistry

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Campylobacter jejuni produces glycoproteins that are essential for virulence. These glycoproteins carry diacetamidobacillosamine (DAB), a sugar that is not found in humans. Hence, the enzymes responsible for DAB synthesis represent potential therapeutic targets. We describe the biochemical characterization of Cj1121c, a putative aminotransferase encoded by the general protein glycosylation locus, to assess its role in DAB biosynthesis. By using overexpressed and affinity-purified enzyme, we demonstrate that Cj1121c has pyridoxal phosphate-and glutamate-dependent UDP-4-keto-6-deoxy-GlcNAc C-4 transaminase activity and produces UDP-4-amino-4,6-dideoxy-GlcNAc. This is consistent with a role in DAB biosynthesis and distinguishes Cj1121c from Cj1294, a homologous UDP-2-acetamido-2,6-dideoxy-␤-L-arabino-4-hexulose C-4 aminotransferase that we characterized previously. We show that Cj1121c can also use this 4-keto-arabino sugar indirectly as a substrate, that Cj1121c and Cj1294 are active simultaneously in C. jejuni, and that the activity of Cj1121c is preponderant under standard growth conditions. Kinetic data indicate that Cj1121c has a slightly higher catalytic efficiency than Cj1294 with regard to the 4-keto-arabino substrate. By site-directed mutagenesis, we show that residues Glu-158 and Leu-131 are not essential for catalysis or for substrate specificity contrary to expectations. We further demonstrate that a cj1121c knock-out mutant is impaired for flagella-mediated motility, for invasion of intestinal epithelial cells, and for persistence in the chicken intestine, clearly demonstrating that Cj1121c is essential for host colonization and virulence. Finally, we show that cj1121c is necessary for protein glycosylation by lectin Western blotting. Collectively, these results validate Cj1121c as a promising drug target and provide the means to assay for inhibitors.

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Structural Studies of FlaA1 from Helicobacter pylori Reveal the Mechanism for Inverting 4,6-Dehydratase Activity

Cited by 49 publications

References 49 publications

Small-Molecule Inhibitors of the Pseudaminic Acid Biosynthetic Pathway: Targeting Motility as a Key Bacterial Virulence Factor

Small-Molecule Inhibitors of the Pseudaminic Acid Biosynthetic Pathway: Targeting Motility as a Key Bacterial Virulence Factor

Identification of ypqP as a New Bacillus subtilis Biofilm Determinant That Mediates the Protection of Staphylococcus aureus against Antimicrobial Agents in Mixed-Species Communities

Cj1121c, a Novel UDP-4-keto-6-deoxy-GlcNAc C-4 Aminotransferase Essential for Protein Glycosylation and Virulence in Campylobacter jejuni

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