Mechanistic Studies on N-Acetylmuramic Acid 6-Phosphate Hydrolase (MurQ) : An Etherase Involved in Peptidoglycan Recycling

Hadi, Timin; Dahl, Ulrike; Mayer, Christoph; Tanner, Martin E.

Mechanistic Studies on N-Acetylmuramic Acid 6-Phosphate Hydrolase (MurQ) : An Etherase Involved in Peptidoglycan Recycling

Dateien

Zu diesem Dokument gibt es keine Dateien.

Datum

2008

Autor:innen

Hadi, Timin

Dahl, Ulrike

Mayer, Christoph

Tanner, Martin E.

DOI (zitierfähiger Link)

https://doi.org/10.1021/bi8014532

Sammlungen

Biologie

Publikationstyp

Zeitschriftenartikel

Publikationsstatus

Published

Erschienen in

Biochemistry. American Chemical Society (ACS). 2008, 47(44), pp. 11547-11558. ISSN 0006-2960. eISSN 1520-4995. Available under: doi: 10.1021/bi8014532

Zusammenfassung

Peptidoglycan recycling is a process in which bacteria import cell wall degradation products and incorporate them back into either peptidoglycan biosynthesis or basic metabolic pathways. The enzyme MurQ is an N-acetylmuramic acid 6-phosphate (MurNAc 6-phosphate) hydrolase (or etherase) that hydrolyzes the lactyl side chain from MurNAc 6-phosphate and generates GlcNAc 6-phosphate. This study supports a mechanism involving the syn elimination of lactate to give an α,β-unsaturated aldehyde with (E)-stereochemistry, followed by the syn addition of water to give product. The observation of both a kinetic isotope effect slowing the reaction of [2-²H]MurNAc 6-phosphate and the incorporation of solvent-derived deuterium into C2 of the product indicates that the C2−H bond is cleaved during catalysis. The observation that the solvent-derived ¹⁸O isotope is incorporated into the C3 position of the product, but not the C1 position, provides evidence of the cleavage of the C3−O bond and argues against imine formation. The finding that 3-chloro-3-deoxy-GlcNAc 6-phosphate serves as an alternate substrate is also consistent with an elimination−addition mechanism. Upon extended incubations of MurQ with GlcNAc 6-phosphate, the α,β-unsaturated aldehydic intermediate accumulates in solution, and 1H NMR analysis indicates it exists as the ring-closed form of the (E)-alkene. A structural model is developed for the Escherichia coli MurQ and is compared to that of the structural homologue glucosamine-6-phosphate synthase. Putative active site acid/base residues are probed by mutagenesis, and Glu83 and Glu114 are found to be crucial for catalysis. The Glu83Ala mutant is essentially inactive as an etherase yet is capable of exchanging the C2 proton of substrate with solvent-derived deuterium. This suggests that Glu83 may function as the acidic residue that protonates the departing lactate.

Fachgebiet (DDC)

570 Biowissenschaften, Biologie

Zitieren

ISO 690

HADI, Timin, Ulrike DAHL, Christoph MAYER, Martin E. TANNER, 2008. Mechanistic Studies on N-Acetylmuramic Acid 6-Phosphate Hydrolase (MurQ) : An Etherase Involved in Peptidoglycan Recycling. In: Biochemistry. American Chemical Society (ACS). 2008, 47(44), pp. 11547-11558. ISSN 0006-2960. eISSN 1520-4995. Available under: doi: 10.1021/bi8014532

BibTex

@article{Hadi2008-11-04Mecha-58634,
  year={2008},
  doi={10.1021/bi8014532},
  title={Mechanistic Studies on N-Acetylmuramic Acid 6-Phosphate Hydrolase (MurQ) : An Etherase Involved in Peptidoglycan Recycling},
  number={44},
  volume={47},
  issn={0006-2960},
  journal={Biochemistry},
  pages={11547--11558},
  author={Hadi, Timin and Dahl, Ulrike and Mayer, Christoph and Tanner, Martin E.}
}

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    <dcterms:abstract xml:lang="eng">Peptidoglycan recycling is a process in which bacteria import cell wall degradation products and incorporate them back into either peptidoglycan biosynthesis or basic metabolic pathways. The enzyme MurQ is an N-acetylmuramic acid 6-phosphate (MurNAc 6-phosphate) hydrolase (or etherase) that hydrolyzes the lactyl side chain from MurNAc 6-phosphate and generates GlcNAc 6-phosphate. This study supports a mechanism involving the syn elimination of lactate to give an α,β-unsaturated aldehyde with (E)-stereochemistry, followed by the syn addition of water to give product. The observation of both a kinetic isotope effect slowing the reaction of [2-&lt;sup&gt;2&lt;/sup&gt;H]MurNAc 6-phosphate and the incorporation of solvent-derived deuterium into C2 of the product indicates that the C2−H bond is cleaved during catalysis. The observation that the solvent-derived &lt;sup&gt;18&lt;/sup&gt;O isotope is incorporated into the C3 position of the product, but not the C1 position, provides evidence of the cleavage of the C3−O bond and argues against imine formation. The finding that 3-chloro-3-deoxy-GlcNAc 6-phosphate serves as an alternate substrate is also consistent with an elimination−addition mechanism. Upon extended incubations of MurQ with GlcNAc 6-phosphate, the α,β-unsaturated aldehydic intermediate accumulates in solution, and 1H NMR analysis indicates it exists as the ring-closed form of the (E)-alkene. A structural model is developed for the Escherichia coli MurQ and is compared to that of the structural homologue glucosamine-6-phosphate synthase. Putative active site acid/base residues are probed by mutagenesis, and Glu83 and Glu114 are found to be crucial for catalysis. The Glu83Ala mutant is essentially inactive as an etherase yet is capable of exchanging the C2 proton of substrate with solvent-derived deuterium. This suggests that Glu83 may function as the acidic residue that protonates the departing lactate.</dcterms:abstract>
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