Tyrosine Latching of a Regulatory Gate Affords Allosteric Control of Aromatic Amino Acid Biosynthesis

Penelope J Cross,Renwick C.J Dobson,Mark L Patchett,Emily J Parker

doi:10.1074/jbc.m110.209924

Abstract

The first step of the shikimate pathway for aromatic amino acid biosynthesis is catalyzed by 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS). Thermotoga maritima DAH7PS (TmaDAH7PS) is tetrameric, with monomer units comprised of a core catalytic (β/α)(8) barrel and an N-terminal domain. This enzyme is inhibited strongly by tyrosine and to a lesser extent by the presence of phenylalanine. A truncated mutant of TmaDAH7PS lacking the N-terminal domain was catalytically more active and completely insensitive to tyrosine and phenylalanine, consistent with a role for this domain in allosteric inhibition. The structure of this protein was determined to 2.0 Å. In contrast to the wild-type enzyme, this enzyme is dimeric. Wild-type TmaDAH7PS was co-crystallized with tyrosine, and the structure of this complex was determined to a resolution of 2.35 Å. Tyrosine was found to bind at the interface between two regulatory N-terminal domains, formed from diagonally located monomers of the tetramer, revealing a major reorganization of the regulatory domain with respect to the barrel relative to unliganded enzyme. This significant conformational rearrangement observed in the crystal structures was also clearly evident from small angle X-ray scattering measurements recorded in the presence and absence of tyrosine. The closed conformation adopted by the protein on tyrosine binding impedes substrate entry into the neighboring barrel, revealing an unusual tyrosine-controlled gating mechanism for allosteric control of this enzyme.

Highlights

Between phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P) to form 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) (Fig. 1)
We demonstrate unequivocally that this N-terminal domain is directly responsible for allosteric control of the enzyme and illuminate a remarkable mechanism of allosteric control in which binding of tyrosine induces a major conformational change inactivating TmaDAH7PS
TmaDAH7PS Is Inhibited by Phenylalanine or Tyrosine— The catalytic activity of wild-type TmaDAH7PS is sensitive to the presence of aromatic amino acids

Summary

EXPERIMENTAL PROCEDURES

Bacterial Strains, Plasmids, and Protein Purification—Standard methodologies were used to amplify the TmaDAH7PS gene (locus tag TM0343) from T. maritima MSB8 (DSM 3109) purified genomic DNA as template and ligate the PCR product into pT7-7 [21]. The gene encoding truncated DAH7PS was amplified from wild-type plasmid and ligated into the pENTR/TEV/dTOPO vector using the Gateway cloning technology (Invitrogen). For truncTmaDAH7PS, initial screens were set up using the sittingdrop vapor diffusion method with droplets of protein solution (0.15 ␮l, 5 mg mlϪ1 in 10 mM BTP, 100 mM KCl, 200 ␮M PEP, pH 7.5) and reservoir solution (0.15 ␮l). For crystallization trials of TmaDAH7PS with tyrosine, initial screens were set up with droplets of 0.15 ␮l of protein solution (10 mg mlϪ1 in 10 mM BTP, 100 mM KCl, 200 ␮M PEP, pH 7.5, 1 mM tyrosine) and 0.15 ␮l of reservoir solution. Data collection and refinement statistics for truncTmaDAH7PS and TmaDAH7PS-tyrosine complex (molecular replacement) truncTmaDAH7PS

Data collection

RESULTS

DISCUSSION