Structure of Pisum sativum Rubisco with bound ribulose 1,5-bisphosphate

Abstract

The first structure of a ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from a pulse crop is reported. Rubisco was purified from Pisum sativum (garden pea) and diffraction-quality crystals were obtained by hanging-drop vapour diffusion in the presence of the substrate ribulose 1,5-bisphosphate. X-ray diffraction data were recorded to 2.20 Å resolution from a single crystal at the Canadian Light Source. The overall quaternary structure of non-activated P. sativum Rubisco highlights the conservation of the form I Rubisco hexadecameric complex. The electron density places the substrate in the active site at the interface of the large-subunit dimers. Lys201 in the active site is not carbamylated as expected for this non-activated structure. Some heterogeneity in the small-subunit sequence is noted, as well as possible variations in the conformation and contacts of ribulose 1,5-bisphosphate in the large-subunit active sites. Overall, the active-site conformation most closely correlates with the `closed' conformation observed in other substrate/inhibitor-bound Rubisco structures.

Figure 1

Preparation of diffracting crystals of PsRubisco. (a) Purification of PsRubisco. SDS–PAGE showing ammonium sulfate (AS) fractions. The 40% fraction contained the highest proportion of Rubisco and was used directly for crystallization after dialysis to remove AS. L, large subunit; S, small subunit. (b) Crystals of PsRubisco. The approximate dimensions of the PsRubisco crystals were 50 × 50 × 20 µm.

Figure 2

PsRubisco maintains the most common hexadecameric Rubisco form. (a) The refined subunits comprising the L₄S₄ asymmetric unit viewed along the fourfold symmetry axis. L subunits are shown in blue and S subunits are shown in red. (b) The L₈S₈ biologically relevant hexadecameric complex viewed along the twofold symmetry axis. The L-subunit symmetry mates are shown in grey, with the asymmetric unit refined L subunits shown in blue and both refined and symmetry-related S subunits shown in red.

Figure 3

The catalytic site of PsRubisco is located at the interface of interacting L subunits. An L₂S₂ unit is viewed, showing two active sites with bound RuBP substrate at the interface of the two twofold symmetry-related L subunits. The expanded image shows detail of the loops from both L-subunit symmetry mates comprising the catalytic site. Loops that undergo conformational variation in the ‘open’ versus ‘closed’ forms of the enzyme are labelled. The L-subunit symmetry mate is shown in grey, with the asymmetric unit refined L subunit shown in blue and both refined and symmetry-related S subunits shown in red. The substrate RuBP is shown with pink phosphate, red O and yellow C atoms.

Figure 4

Details of the catalytic site. (a) A representation of RuBP in the active site of non-activated PsRubisco. (b) Comparison of non-activated RuBP-bound PsRubisco with Ca²⁺-activated inactive RuBP-bound spinach Rubisco. The substrate-binding channel in the PsRubisco structure is shown as a grey surface. Loop 6 of the PsRubisco L subunit (green loop) is folded over and pinned down by the overlapping Lys128 residue (grey surface), yielding a ‘closed’ active-site conformation. This is in contrast to the ‘open’ conformation observed for spinach Rubisco when activated in the presence of Ca²⁺ with RuBP bound (PDB entry 1rxo), which shows loop 6 (yellow loop) folded back and away from the channel. In both panels, discrete atoms are shown with pink phosphate, red O, yellow C and blue N atoms.