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In the phenylalanine biosynthesis of Corynebacterium glutamicum,
DAHP synthase (DS), chorismate mutase and prephenate dehydratase
(PD) are feedback regulated by different aromatic amino acids
including phenylalanine, tyrosine and tryptophan. In the
previous study, they found that partial purified DS from C.
glutamicum is synergistically feedback inhibited by tyrosine and
phenylalanine while this inhibition is released in its mFP-
resistant mutants. They also found that the feedback resistant
phenotype is resulted from the mutation of the PD other than the
DS. These data strongly suggests that the feedback resistant PD
may interacted with DS to cause the release of synergistic
feedback inhibition of DS.Enzyme tagged with six consecutive
histidyl residues and immobilized metal affinity chromatography
(IMAC) were used to study the DS-PD interaction. Purified DS
tagged with 6xHis was incubated with crude cell extract of C.
glutamicum LS1183 harboring wild-type pheA gene and then
subjected to the IMAC. We found that the PD was retained on the
column and asssociated with 6xHis-tagged DS. The results was
also confirmed by immunoblotting against DS and PD polyclonal
antibodies. The interaction between DS and PD is specific
binding and the major interaction forces may not be ionic
binding because the associated PD can not be washed out by high
salt buffer. The interaction between DS and PD was also
confirmed by E. coli two-plasmid system and Far-Western
blotting. However, The real physiological role of DS-PD
interaction is still unclear.
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