Structural mechanism of RuBisCO activation by carbamylation of the active site lysine

Abstract

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a crucial enzyme in carbon fixation and the most abundant protein on earth. It has been studied extensively by biochemical and structural methods; however, the most essential activation step has not yet been described. Here, we describe the mechanistic details of Lys carbamylation that leads to RuBisCO activation by atmospheric CO(2). We report two crystal structures of nitrosylated RuBisCO from the red algae Galdieria sulphuraria with O(2) and CO(2) bound at the active site. G. sulphuraria RuBisCO is inhibited by cysteine nitrosylation that results in trapping of these gaseous ligands. The structure with CO(2) defines an elusive, preactivation complex that contains a metal cation Mg(2+) surrounded by three H(2)O/OH molecules. Both structures suggest the mechanism for discriminating gaseous ligands by their quadrupole electric moments. We describe conformational changes that allow for intermittent binding of the metal ion required for activation. On the basis of these structures we propose the individual steps of the activation mechanism. Knowledge of all these elements is indispensable for engineering RuBisCO into a more efficient enzyme for crop enhancement or as a remedy to global warming.

Fig. 1.

(A) General view of hexadecameric structure G. sulphuraria RuBisCO with three L2S2 dimers visible. The dimers on both sides are in a Cα representation colored by the temperature factors. The front-facing dimer is depicted in a surface representation colored by the polarity of the electrostatic field. Two large, positively charged patches (in blue) indicate the entry to the active site cavity that is lined with highly mobile loops with higher temperature factors coming from the β-barrel catalytic domains and the N-terminal domain (visible in dimers on both sides). These positively charged patches are visible because the disordered N- and C-terminal fragments were not included in our model (not visible in ED). They are responsible for sealing off the active sites during the catalytic cycle. (B) A L2S2 dimer of G. partita RuBisCO in the same orientation as in A with the highlighted N-terminal (orange) and C-terminal (red) regions that are disordered in G. sulphuraria structure. (C) Close-up of the L1 subunit with N- and C-terminal tails marked and the loops numbered. Both termini play a crucial role in stabilizing close conformation of the catalytically competent enzyme. The C-terminal tail stabilizes loops 6, 7, and 5 whereas the N-terminal region stabilizes loop 1 (containing the KPK motif) and loops 2s and 3s of the small domain.

Fig. 2.

The bond-stick model of the nitrosylated Cys460 covered with the omit map electron density (3 σ).

Fig. 3.

The stereo representation of the active site of the enzyme (in bond-stick). (A) The apo-enzyme with O₂ bound at the C-terminal end of the inner β-barrel constituting the active site. The 2Fo-Fc electron density contoured at the 1 σ level is in slate blue, whereas the two omit maps for individual oxygen atoms in the O₂ molecule are contoured at the 4 σ level (purple and red). (B) The active site of the preactivation complex with Mg²⁺ surrounded by three water molecules and the CO₂ molecule. The 2Fo-Fc ED map (contoured at the 1 σ level) is in slate blue; the omit maps, calculated with oxygen atoms omitted from the CO₂ molecule (in red), are contoured at 4 σ; the omit map for Mg²⁺ (in green) is contoured at the 6 σ level; and the omit maps for the surrounding water molecules are contoured at 3.5 σ (in cyan).

Fig. 4.

The ribbon model of the central catalytic domain with bound gaseous ligands at the active site and the accessible surface colored by the electrostatic potential. The ligands are embedded in a positively charged cavity (blue) located at the C-terminal end of the β-barrel of the catalytic domain closed from the top with a negatively charged lid (red) formed by the N-terminal domain of the L subunit of the opposite dimer. Both of these features are responsible for a strong gradient of the electrostatic field across the active site that interacts with quadrupole moments of the gaseous ligands. The bound ligands are (A) dioxygen (in red) and (B) carbon dioxide (in purple).