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N-acetylmuramic acid recognition by MurK kinase from the MurNAc auxotrophic oral pathogen Tannerella forsythia
Aleksandra Cecylia Stasiak1, Karolin Gogler1, Marina Borisova2
1Interfaculty Institute of Biochemistry, University of Tuebingen, Tuebingen, Germany.
Abstract:
The bacterial cell wall consists of a three-dimensional peptidoglycan layer, composed of peptides linked to the sugars N-acetylmuramic acid (MurNAc) and GlcNAc. Unlike other bacteria, the pathogenic Tannerella forsythia, a member of the red complex group of bacteria associated with the late stages of periodontitis, lacks biosynthetic pathways for MurNAc production and therefore obtains MurNAc from the environment. Sugar kinases play a crucial role in the MurNAc recycling process, activating the sugar molecules by phosphorylation. In this study, we present the first crystal structures of a MurNAc kinase, called murein sugar kinase (MurK), in its unbound state as well as in complexes with the ATP analog β-γ-methylene adenosine triphosphate (AMP-PCP) and with MurNAc. We also determined the crystal structures of K1058, a paralogous MurNAc kinase of T. forsythia, in its unbound state and in complex with MurNAc. We identified the active site and residues crucial for MurNAc specificity as the less bulky side chains of S133, P134, and L135, which enlarge the binding cavity for the lactyl ether group, unlike the glutamate or histidine residues present in structural homologs. In establishing the apparent kinetic parameters for both enzymes, we showed a comparable affinity for MurNAc (Km 180 μM and 30 μM for MurK and K1058, respectively), with MurK being over two hundred times faster than K1058 (Vmax 80 and 0.34 μmol min-1 mg-1, respectively). These data might support a structure-guided approach to development of inhibitory MurNAc analogs for pathogen MurK enzymes.
Insights
Tannerella forsythia obtains N-acetylmuramic acid (MurNAc) from its environment. This study reveals crystal structures of MurNAc kinases (MurK and K1058), detailing their active sites and kinetic properties for potential drug development.
Area of Science:
- Biochemistry
- Structural Biology
- Microbiology
Background:
- The bacterial cell wall's peptidoglycan layer is crucial for structure and integrity.
- Tannerella forsythia, a pathogen linked to periodontitis, uniquely acquires N-acetylmuramic acid (MurNAc) due to lacking biosynthetic pathways.
- Sugar kinases are vital for MurNAc recycling by phosphorylating MurNAc.
Purpose of the Study:
- To elucidate the structural and functional characteristics of MurNAc kinases (MurK and K1058) from Tannerella forsythia.
- To identify key residues responsible for MurNAc specificity and enzyme activity.
- To provide a structural basis for developing inhibitors targeting pathogen MurK enzymes.
Main Methods:
- X-ray crystallography was used to determine the structures of MurK and K1058 in various states (unbound, with AMP-PCP, with MurNAc).
- Kinetic parameters (Km and Vmax) were measured for both enzymes.
- Analysis of active site residues was performed to understand MurNAc binding specificity.
Main Results:
- The crystal structures of MurK and K1058, including complexes with ligands, were determined.
- Specific active site residues (S133, P134, L135) were identified as crucial for MurNAc specificity in T. forsythia.
- MurK exhibited significantly higher catalytic activity (Vmax) than K1058, while both showed comparable affinity for MurNAc.
Conclusions:
- The structural and kinetic data provide insights into the function of MurNAc kinases in T. forsythia.
- The identified active site features offer a foundation for designing specific inhibitors against MurK enzymes.
- This research supports a structure-guided strategy for developing novel anti-periodontitis therapeutics targeting bacterial MurNAc metabolism.
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