Effect of Site –Directed Mutagenesis on PS & DHPS

Anna Tartakover, Adina Stoia, and Brian P. Nichols, University of Illinois-Chicago, Chicago, IL, 60607

P- Aminobenzoate is synthesized in bacteria from chorismate and utilized for the synthesis of the vitamin folic acid. Sulfonamides compete with pAB and prevent folic acid synthesis by bacteria, resulting in bacteria death. Because sulfonamides have been used for so many years, we have investigated the function of the pAB biosynthetic enzyme (PS) and the utilization enzyme (DHPS) by site directed mutagenesis of the genes encoding them.

We have chosen to make two different amino acid substitutions in E. coli PS. Asp 299 and Met 305 are highly conserved in PS amino acid sequences. One of the single mutations resulted in a Met->Lys substitution at amino acid position 305 while another mutation resulted in Asp->Lys substitution at amino acid position 299. The function of enzymes with these substitutions is currently being tested and will be compared to identify the significance of these amino acids in the wild type enzyme.
Similarly we substituted amino acid 64 in DHPS and tested for resistance to sulfathiazole. We tested it by introducing a single point mutation. The result was a mutated bacteria with an increased resistance to sulfanilamides of up to 75ug/ml. This new mutant will now be helpful in studying its resistance mechanism and eventually will be compared with that of other organisms, namely pneumococcus.