1,6-Anhydro-N-acetylmuramic acid kinase (AnmK) is a homodimeric enzyme that can convert 1,6-Anhydro-N-acetylmuramic acid into N-acetylglucosamine-6-phosphate by consuming one molecule of ATP in murein recycling in Gram-negative bacteria. Structural data indicate that each subunit of AnmK is comprised of two domains that are separated by a deep active site cleft, which bears similarity to the ATPase core of proteins belonging to the hexokinase-hsp70-actin superfamily of proteins. These data also show that binding of ATP analogue changes the structure of AnmK. Our data suggest that AnmK is an allosteric enzyme and ATP binding follows Monod-Wyman-Changeux model (MWC). The apo-AnmK has two different conformations, one is more open (R state) and the other one is closed (T state). Binding of ATP to AnmK stabilizes the more open (R state) and makes AnmK prone to bind to anhMurNAc sugar.

1,6-Anhydro-N-acetylmuramic acid kinase (AnmK) is a homodimeric enzyme that can convert 1,6-Anhydro-N-acetylmuramic acid into N-acetylglucosamine-6-phosphate by consuming one molecule of ATP in murein recycling in Gram-negative bacteria. Structural data indicate that each subunit of AnmK is comprised of two domains that are separated by a deep active site cleft, which bears similarity to the ATPase core of proteins belonging to the hexokinase-hsp70-actin superfamily of proteins. These data also show that binding of ATP analogue changes the structure of AnmK. Our data suggest that AnmK is an allosteric enzyme and ATP binding follows Monod-Wyman-Changeux model (MWC). The apo-AnmK has two different conformations, one is more open (R state) and the other one is closed (T state). Binding of ATP to AnmK stabilizes the more open (R state) and makes AnmK prone to bind to anhMurNAc sugar.

<p>Bacteria recycle pre-existing peptidoglycan in order to minimize the de novo synthesis of peptidoglycan precursors. The recycling pathway is under study for its chemotherapeutic target potential. Anhydromuramic acid kinase (AnmK) is part of this recycling pathway and catalyzes the dual hydrolysis/phosphorylation of anhMurNAc to MurNAc-6-P. This enzyme has been discovered and introduced, but only minimally characterized. Therefore, the overarching goal of this work was to clone, express and purify AnmK to homogeneity; perform further kinetic characterization; solve the open, closed and transition state mimic-bound conformations of AnmK by x-ray crystallography; and develop a putative mechanism based on the accumulated research findings and <super>18</super>O-labeling studies.</p><p>The anmK gene was successfully cloned as a hexahistidine fusion protein and overexpression was optimized. After exhaustive trials, a final purification scheme was designed to yield homogeneous AnmK in three chromatographic steps and in less than 36 hours. Additionally, the synthesis of both anhMurNAc and a pseudosubstrate (anhGlcNAc) were carried out in 35% and 77% overall yield, respectively. The synthesis of these compounds allowed for both kinetic characterization and structural studies. </p><p>To this end, the structure of de novo AnmK was solved using SAD and high-resolution (1.9 &Aring;) data. Also, an ATP analog (ANP) and anhMurNAc substrate-bound, closed conformation structure (1.95 &Aring;) was solved. These structures elucidated an 11&deg; domain closure of the enzyme upon substrate binding and also revealed the active site geometry to be used to determine potential molecular determinants of specificity. </p><p>Insights into the kinetic mechanism of AnmK were then gathered using multiple techniques. First, the structure of AnmK (2.5 &Aring;) was solved the with a known transition state analog, the MgADP-vanadate complex. Following this structure, which sheds light on the potential importance of a residue other than the catalytic base (Asp187), isotopic labeling was performed with H₂<super>18</super>O. Using NMR and MS, the regiochemical selectivity of AnmK hydrolysis to impart the solvent derived oxygen at C1 was established. Additionally, this was carried out with stereochemical preference to create the &alpha;-anomer of the carbohydrate product. This regiochemistry and stereospecificity drove the design of our putative concomitant hydrolysis/phosphorylation mechanism but we are not able to rule out the formation of a transient phosphoenzyme intermediate.</p><p>This research can be applied to the immediate goal of understanding the function of a single, novel enzyme with unique chemistry and the clarification of the AnmK mechanism will facilitate future investigation into enzymes with dual hydrolase/kinase functionality. Furthermore, this research contributes to understanding of the complex bacterial peptidoglycan layer in order to harness new ideas for developing antibiotics.</p> / Dissertation