NagS is a novel GlcNAc-6P dehydratase.
<p><b>(a)</b> Sensitivity of <i>S. coelicolor</i> mutants to GlcNAc. Spores (5 × 10<sup>5</sup> CFU) of <i>S. coelicolor</i> M145 and its mutant derivatives ∆<i>nagB</i>, SMA11, ∆<i>nagB</i>∆<i>nagS</i>, ∆<i...
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2025
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| Achoimre: | <p><b>(a)</b> Sensitivity of <i>S. coelicolor</i> mutants to GlcNAc. Spores (5 × 10<sup>5</sup> CFU) of <i>S. coelicolor</i> M145 and its mutant derivatives ∆<i>nagB</i>, SMA11, ∆<i>nagB</i>∆<i>nagS</i>, ∆<i>nagB</i>∆<i>nagS</i><sup>C</sup> (∆<i>nagB</i>∆<i>nagS</i> expressing <i>nagS</i>) and ∆<i>nagB</i>∆<i>nagS</i><sup>E</sup> (∆<i>nagB</i>∆<i>nagS</i> with empty plasmid pSET152) were streaked on MM agar plates with 1% mannitol (Mann) and 1% mannitol plus 10 mM GlcNAc (GlcNAc). Strains were cultured for 72 h at 30 °C. Note that <i>nagB</i> mutants can grow in the presence of 10 mM GlcNAc only when SCO4393 (<i>nagS)</i> has been mutated (suppressor SMA11) or deleted. <b>(b)</b> NagS and its role in GlcNAc sensing. Spores of M145 and ∆<i>nagS</i> were plated on MM and R5 with 0, 0.001, 0.01, 0.1, 1, 5, 10, 20, 50, 100, 150, and 200 mM GlcNAc. Note that <i>nagS</i> mutants hardly respond to GlcNAc. <b>(c)</b> Phylogenetic tree of several different types of sugar isomerase (SIS) domain enzymes in bacteria, including NagS, Glucose-6P isomerase (Pgi), Phosphoheptose isomerase (GmhA), <i>N</i>-acetylmuramic acid-6-phosphate etherase (MurQ), Glutamine-fructose-6-phosphate aminotransferase (GlmS), and putative D-galactosamine-6-phosphate deaminase (Agas). The phylogenetic tree was made by MEGA11 (Neighbour-joining method) and built based on the alignment of the amino acid sequences. <b>(d)</b> ITC analysis of NagS with <i>N</i>-acetyl-6-phosphate amino sugar metabolites, namely GlcNAc-6P, ManNAc-6P, and GalNAc-6P. Both GlcNAc-6P and ManNAc-6P are bound well by NagS, while the enzyme did not bind to GalNAc-6P. (<b>e and f</b>) confirmation of NagS products by LC-MS. Extracted ion chromatograms for GlcNAc-6-P (black trace) and its dehydrated product (pink trace) in the enzymatic reaction mixture of GlcNAc-6-P with either active NagS (e) or heat-inactivated NagS <b>(f)</b>. Peaks relating to the product (<i>m</i>/<i>z</i> = 282.0387) are indicated by red arrows. <b>(g)</b> Kinetics of NagS in the presence of 6-PG (0, 0.33, 1 mM). The <i>V</i><sub>0</sub> data used in d were plotted against the substrate concentration, and each assay was performed in triplicate and expressed as a mean ± standard error. The data underlying this Figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3003514#pbio.3003514.s022" target="_blank">S1 Data</a>.</p> |
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