Oligonucleotide-directed mutagenesis of an active site residue in the β-subunit of tryptophan synthase from Salmonella typhimurium
Author:Syndy Rielle Malit
Mentor:Lisa Shamansky, Professor of Chemistry and Biochemistry, California State University San Bernardino
Author: Syndy Rielle Malit, California State University San Bernardino Mentor: Lisa Shamansky, Department of Chemistry and Biochemistry, California State University San Bernardino Tryptophan synthase is a pyridoxal phosphate-dependent α2β2 bienzyme that catalyzes the last two steps of tryptophan synthesis in bacteria, yeasts, mold, plants, and some protozoans. The absence of a synthetic pathway for L-tryptophan in higher animals and in humans makes tryptophan synthase a potential target for development of herbicides and antibiotics. To contribute to the understanding of the catalytic mechanism of tryptophan synthase, site-directed mutagenesis was performed to replace the Serine residue at position 377 of the β-subunit with an Aspartic acid residue. This mutation should allow more detailed investigation of the way tryptophan synthase interacts with the pyridoxal phosphate (PLP) cofactor. A multicopy expression vector, pEBA-10, that encodes the trpA and trpB genes of Salmonella typhimurium was used as the template DNA for megaprimer PCR. Oligonucleotide primers were designed to incorporate the desired amino acid substitution. TA cloning was used to introduce the mutated PCR fragment into plasmid pCRII followed by transformation into competent JM109 cells. Subsequent blue/white screening identified clones whose mutation was verified by dideoxy sequencing. After cutting out the mutated PCR fragment from pCRII, it was ligated into pEBA-10 in place of the corresponding wild type segment and was then used to transform E. coli CB149 cells that lack the Trp operon. These E. coli cells will be induced to express the mutated tryptophan synthase enzyme. This will allow the role of Serine-377 in the acid-base chemistry of the active site pyridoxal phosphate to be investigated at the atomic level using nuclear magnetic resonance crystallography.