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Evidence for the shikimate‐3‐phosphate interacting site in the N‐terminal domain of 5‐enolpyruvyl shikimate‐3‐phosphate synthase of Bacillus subtilis
Author(s) -
Selvapandiyan Angamuthu,
Ahmad Suhail,
Majumder Kumud,
Arora Naresh,
Bhatnagar Raj K.
Publication year - 1996
Publication title -
iubmb life
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.132
H-Index - 113
eISSN - 1521-6551
pISSN - 1521-6543
DOI - 10.1080/15216549600201193
Subject(s) - shikimate pathway , bacillus subtilis , biochemistry , site directed mutagenesis , aspartic acid , mutant , aromatic amino acids , chemistry , amino acid , enzyme , stereochemistry , biology , bacteria , genetics , gene
The role of basic amino acid residues that are highly conserved in the N‐terminal domain of 5‐enolpyruvyl shikimate‐3‐phosphate synthase (EPSPs) in the binding of the substrate, shikimate‐3‐phosphate, has been assessed. Lys 19 and Arg 24 in the Bacillus subtilis EPSPs were substituted by glutamic acid and aspartic acid residues respectively by site‐directed mutagenesis. Native and the mutant proteins were expressed using a two‐vector system and the expressed proteins were purified to near homogeniety. The replacement of either Lys 19 or Arg 24 with a negatively charged residue nearly completely abolished the enzyme activity. The kinetic characterization of the purified wild type and the mutant proteins revealed that the substitution of positively charged residues in the N‐terminal domain (K19 and R24) results in reduced affinity for shikimate‐3‐phosphate (S3P). The results suggest the involvement of these residues in the binding of S3P during enzyme catalysis.