An insight into activation mechanism of plant RuBisCO based on a structure-activity relationship study of His294 using RuBisCO-like protein from Bacillus subtilis

Abstract

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and RuBisCO-like protein (RLP) from Bacillus subtilis catalyze mechanistically similar enolizations. Both enzymes require the activation to be catalytically competent. In the activation, the N^ε of K201 at the active site is carbamylated by non-substrate CO₂ to form carbamate, which is stabilized by the catalytically essential Mg²⁺. Interestingly, RLP shows higher activation level under low CO₂ condition (approximately 2μM) than RuBisCO. We focused on H294 completely conserved in RuBisCO and RLPs, because this residue is probably involved in stabilization of carbamate through a hydrogen bonding between N^ε of H294 and the carbamate oxygen (2.8A) in RLP. H294Q, H294N and H294A RLPs had 92-98% decreases in k_cat, 2.2- to 3.0-fold increases in K_m. All mutants required higher CO₂ concentration for maximal activities compared to wild-type. These results suggested that H294 stabilizes the carbamate in RLP. By contrast, H294 is far from the carbamate oxygen (3.4A) in plant RuBisCO, resulting in the destabilization of carbamate. Alternatively, this may enable to regulate the RuBisCO activity by activation in plants.