Open AccessSerine 187 is a crucial residue for allosteric regulation of Corynebacterium glutamicum 3‐deoxy‐ D ‐arabino‐heptulosonate‐7‐phosphate synthase
Author(s) -
Liao HuiFen,
Lin LongLiu,
Chien Hungchien Roger,
Hsu WenHwei
Publication year - 2001
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2001.tb09446.x
Subject(s) - corynebacterium glutamicum , biochemistry , enzyme , tyrosine , atp synthase , site directed mutagenesis , allosteric regulation , biology , biosynthesis , mutant , escherichia coli , chemistry , gene
Corynebacterium glutamicum 3‐deoxy‐ D ‐arabino‐heptulosonate‐7‐phosphate (DAHP) synthase is sensitive to feedback inhibition by tyrosine. One feedback‐insensitive mutant was obtained by in vitro chemical mutagenesis and the mutation was identified as a C→G mutation at nucleotide 560 causing a Ser 187 to Cys 187 substitution. Replacing Ser 187 with cysteine, tyrosine or phenylalanine by site‐directed mutagenesis not only reduced the enzymatic activity but also relieved its feedback inhibition by tyrosine, while Ser 187 Ala exhibited a comparable activity to that of wild‐type enzyme and sensitized to allosteric regulation. The His 6 ‐tagged enzymes were expressed in Escherichia coli and purified to homogeneity by immobilized nickel‐ion affinity chromatography. Kinetic analysis showed that tyrosine is a competitive inhibitor of phosphoenol pyruvate, one of the precursors for DAHP biosynthesis.