Tyrosine latching of a regulatory gate affords allosteric control of aromatic amino acid biosynthesis

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.

FIGURE 1.

Reaction catalyzed by DAH7PS.

FIGURE 2.

Response of TmaDAH7PS activity to increasing concentrations of tyrosine (■), phenylalanine (○), and tryptophan (▴). Assays contained E4P (180 μm), PEP (123 μm), MnSO₄ (100 μm), and 2 μg of TmaDAH7PS in 50 mm BTP (pH 7.3).The data points were measured in triplicate. Error bars indicate S.D.

FIGURE 3.

Structure of ^truncTmaDAH7PS. A, ^truncTmaDAH7PS monomer (red) overlaid with wild-type TmaDAH7PS (1RZM) monomer (blue with the regulatory domain in gray) (r.m.s.d. = 0.33 Å). B, ^truncTmaDAH7PS dimer (red). C, ^truncTmaDAH7PS dimer (red) overlaid with TmaDAH7PS tetramer (blue with the regulatory domain in gray) (r.m.s.d. = 0.73 Å).

FIGURE 4.

Structure of TmaDAH7PS co-crystallized with tyrosine. A, liganded tetramer looking down on, and in line with, the tetramer plane. B, interactions of regulatory domains of diagonally opposite molecules of tetrameric TmaDAH7PS; tyrosine is shown as cyan sticks. C, electron density (2F_o − F_c, contoured at 1σ (blue); F_o − F_c, contoured to 3σ (green); F_o − F_c, contoured to −3σ (red)) showing tyrosine binding between the regulatory domains of opposing monomers. D, stereo view of tyrosine-binding site. Residues contributed from different monomer regulatory domains are represented by either magenta or blue.

FIGURE 5.

Comparison of tyrosine-bound and tyrosine-free structures revealing major domain movement on tyrosine binding. A, overlay of tyrosine-bound (blue) and tyrosine-free (green) monomer. B, overlay of tyrosine-bound (blue) and tyrosine-free (green) tetramer looking down on, and in line with, the tetramer plane. C, schematic representation of regulatory domain shift of TmaDAH7PS on tyrosine binding. Tyrosine binding (yellow oval) results in structural reorganization so that the regulatory domains of diagonally opposite molecules occlude the active site (white shading) of the neighboring barrel. Each monomer has the barrel symbolized as a circle and the regulatory domain symbolized as a triangle.

FIGURE 6.

SAXS measurements of TmaDAH7PS, with and without tyrosine. Theoretical scattering profiles were generated from crystallographic coordinates of both the tyrosine-free open structure, 1RZM (dotted line), and the tyrosine-bound (PDB code 3PG9), closed structure (dashed line) using CRYSOL. These theoretical plots were compared with the experimentally determined TmaDAH7PS SAXS data (open circles) in both the absence (A) and the presence (B) of tyrosine. The experimental SAXS data were fitted to a distribution of both open and closed forms using OLIGOMER (line). χ_v² values for the fit to the calculated CRYSOL data for the open (1RZM) and closed (3PG9) conformations and values for the OLIGOMER fit for the data shown in A are 0.48, 11.4 and 0.38, respectively, and the values for B are 11.0, 1.31, and 0.71. Guinier plots and P(r) are shown as supplemental Figs. S10 and S11.