Researcher(s)
- Damian Gonzalvo, Biochemistry, University of Delaware
Faculty Mentor(s)
- Damian Gonzalvo, Department of Chemistry and Biochemistry1, Department of Biological Sciences2, University of Delaware 19717, University of Delaware
Abstract
Tannerella forsythia is a Gram-negative oral pathogen often associated with periodontal diseases; it has also been implicated in other diseases, including cardiovascular disease and Alzheimer’s. Unlike most other bacteria, T. forsythia is unable to synthesize N-acetyl muramic acid (MurNAc), an essential precursor for the synthesis of it peptidoglycan, and therefore it must rely on salvaging fragments of MurNAc from its environment. This unique pathway opens up the possibility for more selective targeting of this pathogen. One method of inhibition can come from the targeting of specific enzymes involved in this pathway. One such enzyme, AmgK, a kinase that catalyzes the phosphorylation of MurNAc into MurNAc-1 phosphate, a crucial step in the incorporation of the salvaged MurNAc into the bacterial cell wall. Structural analysis through Alphafold-2 was able to predict a conserved kinase domain, as well as a unique C-terminal ATP-Bind-2 domain, which is not found in other commonly found AmgK homologs and was proposed to have an RNA-binding domain. The objective of this study was to biochemically characterize AmgK from T. forsythia. Recombinant AmgK was expressed using constructs containing either an N-terminal His₆ tag or a GST affinity tag. The purification conditions were optimized.and analyzed by SDS-PAGE to assess expression levels and purification efficiency. The enzymatic activity of AmgK was characterized using an ADP-coupled kinase assay, and substrate specificity was examined through small-scale chemoenzymatic synthesis reactions with MurNAc and related analogs. Additionally, circular dichroism (CD) spectroscopy was employed to assess the secondary structure and conformational stability of the purified protein. To investigate the proposed RNA-binding properties of the C-terminal domain, purified protein samples were analyzed using SYBR Gold staining following denaturing urea polyacrylamide gel electrophoresis. The presence of co-purifying nucleic acids was evaluated as preliminary evidence of potential RNA association. Although these experiments do not establish direct or sequence-specific RNA binding, the observed results support further investigation of the putative RNA-binding function of the C-terminal domain. Together, these studies provide insights into the biochemical properties, substrate preferences, and potential multifunctional roles of T. forsythia AmgK, laying the foundation for future investigations into MurNAc salvage and its potential as a therapeutic target.