Serine 187 is a crucial residue for allosteric regulation of Corynebacterium glutamicum 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase.
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.
Liao
H F
HF
Institute of Molecular Biology, National Chung Hsing University, Taichung 402, Taiwan.
Lin
L L
LL
Chien
H R
HR
Hsu
W H
WH
D010723:3-Deoxy-7-Phosphoheptulonate Synthase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000217:Acyltransferases / Q000378:metabolism; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D003352:Corynebacterium / Q000201:enzymology* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012694:Serine / Q000737:chemistry / Q000235:genetics; D013403:Sugar Phosphates / Q000378:metabolism*
D013403:Sugar Phosphates; C013469:3-deoxy-D-arabino-heptulosonate-7-phosphate; D012694:Serine; D000217:Acyltransferases; C020051:glycerone-phosphate O-acyltransferase; D010723:3-Deoxy-7-Phosphoheptulonate Synthase