Ultrahigh Resolution Structure of the Heat-stable Form-IAq RuBisCO from the Thermophilic Purple Sulfur Bacterium Thermochromatium tepidum

Abstract

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) catalyzes the initial carbon fixation reaction in the Calvin-Benson-Bassham cycle in plants and most microbial autotrophs. Among the many forms of RuBisCOs, form-I--a protein complex containing 8 large and 8 small subunits--is the most common, representing over 90% of all known RuBisCOs. Although many form-I RuBisCO structures have been determined, no structure has been reported for a form-IAq RuBisCO. Here, and at ultrahigh (1.55 [A]) resolution, we detail the structure of the heat-stable form-IAq RuBisCO from the thermophilic and anaerobic purple bacterium Thermochromatium (Tch.) tepidum. The overall structure of the Tch. tepidum form-IAq RuBisCO resembles both a form-IAc RuBisCO from a chemolithotrophic sulfur bacterium and a synthetic form-I RuBisCO reconstructed from ancestral sequences. However, despite significant structural and sequence similarities with other form-I RuBisCOs, the Tch. tepidum form-IAq RuBisCO shows significantly greater interactions between adjacent small subunits through their extended N-terminal domains that contain a characteristic six-residue insertion unique to form-IAq RuBisCOs. Structural differences of Tch. tepidum RuBisCO from its mesophilic relative Allochromatium vinosum suggests the mechanisms of its enhanced thermostability. In addition to small subunit interactions, key substitutions on the hydrophilic surface of the small subunits likely contribute to its thermostability as well. It has been proposed that form-I RuBisCOs first appeared in anaerobic thermophiles. Our structure of the Tch. tepidum RuBisCO represents the first such structure of a form-IAq enzyme, providing fresh clues for unraveling the evolutionary history of RuBisCO and new details for how this key enzyme remains active at elevated temperatures.