Serine 187 is a crucial residue for allosteric regulation of Corynebacterium glutamicum 3-deoxy- D-arabino-heptulosonate-7-phosphate synthase

Abstract

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 187Ala 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.