Univ., Diss., 2009--Marburg.

<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

Univ., Diss.--Regensburg, 1992.

Conversion of <i>Campylobacter upsaliensis</i> to the nonculturable but viable coccoid form was characterized. Chloramphenicol did not prevent the conversion. Severe decreases in isocitrate dehydrogenase activity and oxygen uptake and extensive degradation of ribosomal RNA suggest that the coccoid form is a degenerative form rather than part of a life cycle. The autolysins of spiral and coccoid forms of <i>C. upsaliensis</i> were also studied. Autolytic activity in the soluble and sediment fractions of sonicates of the spiral and the coccoid form of <i>C. upsaliensis</i> could not be demonstrated by native (nondenaturing) PAGE. Autolysins were detected, however, by using denaturing SDS-PAGE gels containing either purified <i>E. coli </i> peptidoglycan or whole cells of <i>Micrococcus luteus</i> as the turbid substrate, with subsequent renaturation by treatment with Triton X-100 buffer. In renaturing gels that contained <i>E. coli</i> peptidoglycan, 14 autolytic bands were detected ranging from 200 kDa to 12 kDa. In similar gels containing whole cells of <i>M. luteus</i> , only a single band appeared having a molecular weight of 34 kDa. This band corresponded to one of the bands present in the gels containing <i>E. coli </i> peptidoglycan. This common autolysin was isolated by adsorbing it from <i>C. upsaliensis</i> lysates onto <i>M. luteus</i> cells and then subjecting these cells to renaturing SDS-PAGE in gels containing <i>E. coli</i> peptidoglycan. The 34 kDa autolysin differed from a single 51 kDa autolysin unique to the <i>M. luteus cells</i>. The 34 kDa autolysin was isolated from an SDS-PAGE gel and was pure when tested by isoelectric focusing. The N-terminal amino acid sequence analysis showed the first 15 amino acids of the 34 kDa autolysin to have 67% identity with a part of antigenic protein PEB4 of <i>Campylobacter jejuni</i>. The purified autolysin was used to immunize rabbits and the antibodies produced precipitated autolytic activity from cell lysates. The specificity of the antibodies was shown by Western blotting: only a single specific band occurred, with a molecular weight of 34 kDa, and thus it seems unlikely that the 34 kDa autolysin was derived from any of the other autolysins that were detected. / Ph. D.