8 research outputs found
<i>Sp</i>OatA<sub>C</sub> and <i>Sa</i>OatA<sub>C</sub>-catalyzed <i>O</i>-acetyltransferase reactions.
<p>ESI-MS analysis of reaction products of 2 mM chitotetraose (G<sub>4</sub>) in 50 mM sodium phosphate buffer pH 6.5 incubated at 37 <sup>o</sup>C for 1 h in the absence (control) and presence of enzymes (5 μM, final concentration) with 1 mM concentrations of <b>A,</b> acetyl-CoA; <b>B,</b> 4MU-Ac; or <b>C,</b> <i>p</i>NP-Ac as potential donor acetyl substrates.</p
Structural comparison of <i>Sp</i>OatA<sub>C</sub> with representative members of the SGNH/GDSL and AlgX-N/DHHW families of enzymes.
<p><b>A</b>. The cartoon representation of <i>Sp</i>OatA<sub>C</sub> (gray) is superposed with <i>Bos taurus</i> platelet-activating factor acetylhydrolase (PAF-AH) (blue) and the N-terminal catalytic domain of <i>P</i>. <i>aeruginosa</i> AlgX (green). Right inset: Cartoons depicting the respective peptide backbones of the Block II-loop in the three enzymes. <b>B</b>. Sequence alignments of residues comprising the signature sequence Blocks of the SGNH/GDSL and AlgX-N/DHHW families of enzymes. Red lettering denotes invariant residues in the respective families.</p
Specific activities of <i>Sp</i>OatA<sub>C</sub> and <i>Sa</i>OatA<sub>C</sub> variants.
<p>Specific activities of <i>Sp</i>OatA<sub>C</sub> and <i>Sa</i>OatA<sub>C</sub> variants.</p
Stem peptide specificity of <i>Sp</i>OatA<sub>C</sub> and <i>Sa</i>OatA<sub>C</sub>.
<p><b>A</b>. Stacked and offset ESI-mass spectra of mutanolysin-treated products from reactions of 10 μg·mL<sup>-1</sup> of (left to right) muroglyan-5P, muroglycan-4P, and muroglycan-3P in 50 mM sodium phosphate buffer pH 6.5 incubated with 0.5 mM <i>p</i>NP-Ac in the absence (control) and presence of the respective enzyme (10 μM). The major O-acetylated products are labeled in blue which are 42.01 m/z units larger than the respective unmodified PG monomer. <b>B</b>. MS/MS analysis of the major product ions identified in the respective panels above and <b>C</b>, interpretation of the corresponding fragment ions.</p
Activity and domain structure of OatA.
<p><b>A</b>. PG is comprised of alternating GlcNAc (G) and MurNAc (M) residues with stem peptides (small circles). The lysozymes of innate immunity systems (LYZ) hydrolyze the linkage between M and G residues which results in cell rupture and death. OatA O-acetylates the C-6 hydroxyl group of MurNAc residues (red triangles) in PG of pathogenic Gram-positive bacteria which sterically inhibits the action of the lysozymes, thereby conferring resistance to this first line of the innate immune response. <b>B</b>. Domain organization of OatA. This bimodular protein is comprised of two domains, a predicted N-terminal Acyl_transferase_3 (Pfam PF01757) transmembrane domain and a C-terminal SGNH/GDSL extracytoplasmic domain. The genes encoding OatA from <i>S</i>. <i>aureus</i> and <i>S</i>. <i>pneumoniae</i> were engineered to produce the 25 kDa C-terminal SGNH/GDSL domains (OatA<sub>C</sub>) as shown.</p
Active site structure of <i>Sp</i>OatA<sub>C</sub>.
<p>The H-bonding network of catalytic and oxyanion hole residues in <b>A</b>, resting <i>Sp</i>OatA<sub>C</sub> and <b>B</b>, <i>Sp</i>OatA<sub>C</sub> in complex with MeS (<i>Sp</i>OatA<sub>C</sub>-MeS). The water molecule w1 and the potential inter-residue interactions are depicted as a red sphere and black dashed lines, respectively. <b>C</b>. The <i>2F</i><sub><i>o</i></sub><i>-F</i><sub><i>c</i></sub> electron density map of the MeS-Ser438 adduct contoured at 1.0 σ. <b>D</b>. Superposition of the <i>Sp</i>OatA<sub>C</sub> and <i>Sp</i>OatA<sub>C</sub>-MeS active sites.</p
Proposed pathway for PG O-acetylation by OatA.
<p>(1) An acetyl group from an unidentified donor is obtained from the cytoplasm by the N-terminal domain of OatA and then it is translocated across the inner membrane. (2) The extracytoplasmic C-terminal domain of OatA accepts the acetyl group and catalyzes the acetyltransfer to modify the C6-OH of MurNAc residues within PG. (3) The final product: 6-<i>O</i>-acetyl-MurNAc.</p
Proposed mechanism of action of OatA.
<p>(i) An acetyl donor binds into the active site and causes Asn491 to align appropriately within the oxyanion hole. The H-bonding network between the catalytic triad residues increases the nucleophilicity of the Ser438 hydroxyl which attacks the carbonyl center of the bound acetyl group. (ii) The putative transient tetrahedral oxyanion intermediate is stabilized by the backbone amide of Ser438 and side-chain amide of Asn491. (iii) An acetyl-enzyme intermediate forms with the departure of the donor group. (iv) A MurNAc residue on PG binds, possibly involving Asn491, and (v) His571 serves as a base to abstract the proton from the C6 hydroxyl group to permit its nucleophilic attack on the carbonyl center of the acetyl-enzyme. (vi) The second transient oxyanion intermediate formed is stabilized by the oxyanion hole residues prior to its (vii) collapse releasing the 6-<i>O</i>-acetylPG product. Not depicted are the transition states that lead to and from each of the oxyanion intermediates. R, the acetyl donor molecule; R’, GlcNAc residues of a PG glycan chain; R”, lactyl group and associated stem peptide.</p