Mechanistic and structural investigation of Yersinia pestis arginine decarboxylase

Yersinia pestis, the etiological agent of bubonic and pneumonic plague, is a Category A bioterrorism agent, indicating its potential for causing widespread social disruption. Plague infections are treatable with antibiotic therapy; however, resistance has been identified. To combat the development of resistance, novel antimicrobials need to be developed.; The pyridoxal-5'-phosphate (PLP)-dependent polyamine pathway enzyme arginine decarboxylase plays a key role in Y. pestis biofilm formation, a known antibiotic resistance mechanism in bacteria. The mechanistic and structural characteristics of this enzyme were investigated to identify new targets for drug design.; Steady state kinetics and spectroscopic experiments revealed the importance of C529 in regulating the electronic environment surrounding the cofactor. Mutations to this residue caused a decrease in catalytic efficiency for substrate, L-arginine. Further effects are seen in the presence of alternative substrates. It was also noted that steric interference has a more negative effect on the catalytic efficiency of the ADC mutant enzymes than electronic modifications when alterations are made to the guanidinium moiety.; Pre-steady state kinetic methods revealed the formation of two geminal diamine intermediates for ADC in the presence of agmatine, a novel finding for a PLP-dependent enzyme. The relationship between the two species is pH-dependent. The formation of a geminal diamine intermediate was shown to be absolutely dependent on Mg2+.; Several methods were used to improve the quality of X-ray crystallographic data for ADC. Using C529A ADC, it was possible to grow large, reproducible crystals that were able to withstand long periods of time in the X-ray beam. Resolution was improved to 2.2 A by incorporating lanthanides into the protein.; Using members of the fold III PLP-dependent decarboxylase family as templates, a structure of the ADC dimer was produced using molecular modeling. The location and structure of an insertion of ∼100 amino acids was also determined. The active site was modeled with L-arginne bound via an external aldimine bond to PLP, revealing the location and orientation of important active site residues. In addition, ADC was found to have a predominantly negative electronic surface potential, an important feature for an enzyme with a basic substrate like L-arginine.; Keywords. arginine decarboxylase, pyridoxal-5'-phosphate, polyamines, biofilm, antibacterial...