3 - Deoxy-d-arab G Ketone Sugar -7 - The Structure Of Phosphate Synthase Arog Of, And Function Study

In microorganisms and plants the first step in the aromatic acid biosynthetic pathway is the condensation of PEP and E4P, with the release of the phosphate of PEP and the formation of DAHP. This reaction is catalyzed by DAHP synthase, which in microorganisms is also the target for negative-feedback regulation of the pathway mediated either by the pathway intermediate chorismate, or by endproducts phenylalanine, tyrosine and tryptophan. In Escherichia coli, aroG gene encodes phenylalanine-sensitive 3 -deoxy-D-arabino-heptulosonate-7-phosphate synthase isoenzyme AroG that catalyzes the first committed step of shikimate pathway. Here we study the essential amino acid residues involved in the formation of feedback inhibition site of AroG, and the effects of N-terminus on feedback inhibition and its quaternary structure, and the importance of the structural "D2" symmetry to allosteric inhibition.Our work elucidates the formation of the feedback inhibition site. Point mutations reveal that amino acid replacements at Phel44, Leul75, Leul79, Phe209 and Val221 partially or completely relieve the feedback inhibition of AroG addressed by 1 mM phenylalanine. Combined with the 3D structure of AroG, our results demonstrate that the feedback inhibition site consists of Asp6, Asp7, Ile10, Ile13, Pro150, Gln151, Leul75, Leu179, Serl80, Phe209, Ser211and Val221. The pocket accommodating the aromatic ring of phenylalanine is formed predominantly by hydrophobic side-chains, including those of Ile10, Ile13, Prol50, Leul75, Leul79, Phe209, Ser211 and Val221; whilst the amino group and carboxylate group of phenylalanine is coordinated by Asp6, Asp7, Gln151 and Ser180.The N-terminal region is found to be important to the function of AroG. Point mutation reveals that residue substitution of IlelO to Ala10 leads to desensitization in the presence of 1 mM phenylalanine, simultaneously, exhibits a dramatic decrease inenzymatic activity. Asn5Lys also shows a moderate release of the feedback inhibition with comparable specific enzymatic activity to the wild type AroG. These results unambiguously demonstrate that a single amino acid residue substitution is sufficient to generate a phenylalanine-insensitive form of AroG. Furthermore, the mutant A(l-15) lacking the first fifteen N-terminal amino acid residues exhibits no sensitivity toward phenylalanine in a range of 0 ~ 1 mM phenylalanine. However, the enzymatic activity of this mutant decreases by more than 90%. The results of Superose 6 gel-filtration chromatography and Western Blot suggest that the deletion mutant cause a gigantic change in global structure of AroG. The N-terminus truncated mutant can not form tight dimeric and tetrameric structure, and leads to the destruction of the quaternary structure, which is quite the contrary to wild type AroG. These data indicate that N-terminus of AroG is not only involved in the feedback inhibition, but also plays a role in the formation of stable dimer which is an essential structure for its enzymatic activity.According to the 3D structure, "D2" symmetry of AroG, to which contributed by Leu 16, Trp215 and His217, is essential for the feedback inhibition. Point mutations at these residues exhibit partially relief of feedback inhibition in the presence of 1 mM phenylalanine. Leul6Ala, His217Ala and His217Asp exhibit comparable enzymatic activities to the wild type, while Trp215Ala decreases in the enzymatic activity by more than 60%. Double-mutations Trp215Ala/His217Ala and Leul6His/His217Leu cause completely desensitization with no decrease in enzymatic activity. These results suggest that mutations at these residues cause the destruction of the symmetry, and interfere with the signal transmission of feedback regulation, which leads to the partially or completely relief of feedback inhibition. These data implicate that the structural "D2" symmetry is essential conformation for the signal transmission of feedback inhibition.