Analysis of the binding site of NagS and activity of NagS mutants.
<p><b>(a)</b> NagS active site with bound GlcNAc-6P (gray carbons), protein residues are coloured with pale blue and yellow carbons to indicate the 2 monomers forming the active site. 2|Fo|-|Fc| electron density contoured at 1.2 σ is shown as cyan mesh. Hydrogen bonds are indicated...
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2025
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| Sumari: | <p><b>(a)</b> NagS active site with bound GlcNAc-6P (gray carbons), protein residues are coloured with pale blue and yellow carbons to indicate the 2 monomers forming the active site. 2|Fo|-|Fc| electron density contoured at 1.2 σ is shown as cyan mesh. Hydrogen bonds are indicated by dashed yellow lines. <b>(b)</b> GlcNAc-6P binding site of NagS, with hydrogen bonding distances, or distances between hydrogens and hydrogen bond acceptors indicated. The other molecule of the dimer contributes amino acids ArgB64, AlaB65, GlyB227, and AsnB228. The inset indicates the likely electron rearrangements required for ring-opening, the first step of catalysis. <b>(c)</b> Putative transition state after ring-opening, prior to rotations about the C5–C6 and C1–C2 bonds of GlcNAC-6P (indicated in red), that presumably precede subsequent ring closing. These rotations are associated with the rearrangement of hydrogen bonds. This likely requires conformational changes that in the crystal are inhibited by crystal contacts, explaining why the crystals are not enzymatically active. <b>(d)</b> NagS active site with bound 6-phosphogluconate (gray carbons), protein residues are coloured with pale blue and yellow carbons to indicate the 2 monomers forming the active site. 2|Fo|-|Fc| electron density contoured at 1.2 σ is shown as cyan mesh. <b>(e)</b> Hydrogen bonding distances observed in the 6-phosphogluconic-inhibited state of NagS. The inhibited state suggests how GlcNAc-6P rearranges upon ring-opening, and likely reflects the transition state prior to ring closing, which probably involves Ser91, Glu94, and AsnB228. <b>(f)</b> NagS enzyme activity in vivo. GlcNAc sensitivity of ∆<i>nagB</i>∆<i>nagS</i> harboring clones expressing NagS mutants H53A, S54A, R64A, S91A, E94A, S119A, S121A, D179A, or N228A were grown on MM agar supplemented with 1% mannitol (Mann) and 1% mannitol plus 10 mM GlcNAc (GlcNAc). Single colonies are most likely spontaneous suppressors. <b>(g)</b> In vitro activity (<i>V</i><sub>max</sub>) for wild-type NagS (WT) and NagS variants, with the substrate of GlcNAc-6P. 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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