Intersubunit signaling in glutamate-1-semialdehyde-aminomutase

Abstract

Enzymes are highly dynamic and tightly controlled systems. However, allosteric communication linked to catalytic turnover is poorly understood. We have performed an integrated approach to trap several catalytic intermediates in the alpha2-dimeric key enzyme of chlorophyll biosynthesis, glutamate-1-semialdehyde aminomutase. Our data reveal an active-site "gating loop," which undergoes a dramatic conformational change during catalysis, that is simultaneously open in one subunit and closed in the other. This loop movement requires a beta-sheet-to-alpha-helix transition to assume the closed conformation, thus facilitating transport of substrate toward, and concomitantly forming, an integral part of the active site. The accompanying intersubunit cross-talk, which controls negative cooperativity between the allosteric pair, was explored at the atomic level. The central elements of the communication triad are the cofactor bound to different catalytic intermediates, the interface helix, and the gating loop. Together, they form a molecular switch in which the cofactor acts as a central signal transmitter linking the subunit interface with the gating loop.

Scheme 1.

Proposed catalytic mechanism (2, 3). The reaction mechanism starts with the formation of a complex between PMP and GSA (i). After ketimine-5 formation (ii; KE-5), the double bond shifts and the external aldimine between PLP and 5′-DAVA is formed (iii; EA-5), followed by the formation of the internal aldimine between PLP and Lys-273 with reorientation of DAVA (iv; DAVA-IA). The second half of the reaction cycle (v–vii) is the backward reaction. Starting with the external aldimine between PLP and 4′-DAVA (v; EA-4), the ketimine-4 between PMP and ALA is established (vi; KE-4). Finally, ALA in the PMP form of GSAM can be released (vii). The catalytic intermediates marked with an asterisk were determined by x-ray crystallography.

Fig. 1.

Crystal structure of GSAM in the PMP (KE-4)/PLP (DAVA-IA) form. (A) Overall stereo presentation of α2-dimeric GSAM. In subunit A, the N- and C-terminal domains as well as the cofactor binding domain are shown in different blue tones. Subunit B is shown in yellow. Cofactors and catalytic intermediates are highlighted. Both termini are denoted. The gating-loop regions disobeying local 2-fold symmetry and the interface helices (residues 121–138) are shown in blue (open) and red (closed), respectively. The gating loop is located at the dimer interface and extends toward the active site in the closed conformation. (B) Superposition of residues 150–183 in open (light blue/blue) and closed (yellow/red) conformation. Cofactors within the active sites are colored accordingly. The hinge element (Leu-158 and Ser-172) and Ser-163 are denoted. The β-hydroxy group of Ser-163 moves ≈22 Å.

Fig. 2.

Transition of the gating loop between opened, closed, and reopened conformation. (A) View of the opened gating loop from the crystal structure of the double PMP form of GSAM (2). Fixation of DAVA with its 4′-amino group suggests that the gating loop offers a channeling mechanism to transport substrate into the active-site pocket. The backbone helix is suggested to be the anchoring point for GluTR (26). (B) Active-site pocket in the closed gating-loop conformation. Shown is a superposition of intermediate step iii/preparation 3 (stick-and-ball mode and water as red spheres) and step iv/preparation 4 (in yellow). In the external aldimine, the carboxy group fixation of the intermediate is mediated by three water molecules (W1–W3) and Ser-29. In the DAVA-IA state, all hydrogen bonds between the catalytic intermediate and the water molecules are disrupted and W3 is replaced by the carboxy group of DAVA. (C) Active-site pocket in the reopened and disordered gating-loop conformation. Shown is a superposition of step vi/preparation 4 (stick-and-ball mode and water as red spheres) and step iv/preparation 3 (in yellow). Electrostatic interactions of the catalytic intermediate with Tyr-301* and Ser-163 are absent. In KE-4 and EA-5, Glu-406 reveals multiple conformations and the carboxy group of ketimine-4 interacts again with waters W2 and W3. Dotted lines in black and yellow indicate hydrogen bonds in the EA-5/KE-4 and the DAVA-IA states, respectively. Residues marked with an asterisk depict the other subunit.

Fig. 3.

The communication triad and intersubunit cross-talk. (A) Invariant amino acid residues within the PMP/PLP form of GSAM (also see Fig. 5). Residues of the interface helix involved in crossover interactions are highlighted in green and denoted in single-letter code. All waters shown are conserved among the GSAM structures. The diminished minicore of the cofactor is completed by a network of water molecules providing contacts to intermediate and active-site residues Tyr-150 (W1) as well as intersubunit contacts in the second layer (WI1–WI3). (B and C) Cross-talk in the double PLP form of GSAM (Scheme 1, superposition of steps iii and iv) in two different orientations. The EA-5 state is shown in stick-and-ball mode, whereas DAVA-IA is highlighted in cyan. Interactions are marked in dotted lines. Beginning from residue Ala-154, the gating loop of the subunit containing DAVA-IA is disordered.