Evidence for the shikimate-3-phosphate interacting site in the N-terminal domain of 5-enolpyruvyl shikimate-3-phosphate synthase of Bacillus subtilis.
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.
International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
A
A
Selvapandiyan
S
S
Ahmad
K
K
Majumder
N
N
Arora
R K
RK
Bhatnagar
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases; D001412:Bacillus subtilis / Q000201:enzymology*; D001665:Binding Sites; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017510:Protein Folding; D000644:Quaternary Ammonium Compounds / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012765:Shikimic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D014166:Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000644:Quaternary Ammonium Compounds; D011994:Recombinant Proteins; D012765:Shikimic Acid; C042786:shikimic acid-3-phosphate; D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase