Abstract
The Calvin-Benson-Bassham cycle is responsible for carbon dioxide fixation in all plants, algae, and cyanobacteria. The enzyme that catalyzes the carbon dioxide-fixing reaction is ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Rubisco from a hyperthermophilic archaeon Thermococcus kodakarensis (Tk-Rubisco) belongs to the type III group, and shows high activity at high temperatures. We have previously found that replacement of the entire α-helix 6 of Tk-Rubisco with the corresponding region of the spinach enzyme (SP6 mutant) results in an improvement of catalytic performance at mesophilic temperatures, both in vivo and in vitro, whereas the former and latter half-replacements of the α-helix 6 (SP4 and SP5 mutants) do not yield such improvement. We report here the crystal structures of the wild-type Tk-Rubisco and the mutants SP4 and SP6, and discuss the relationships between their structures and enzymatic activities. A comparison among these structures shows the movement and the increase of temperature factors of α-helix 6 induced by four essential factors. We thus supposed that an increase in the flexibility of the α-helix 6 and loop 6 regions was important to increase the catalytic activity of Tk-Rubisco at ambient temperatures. Based on this structural information, we constructed a new mutant, SP5-V330T, which was designed to have significantly greater flexibility in the above region, and it proved to exhibit the highest activity among all mutants examined to date. The thermostability of the SP5-V330T mutant was lower than that of wild-type Tk-Rubisco, providing further support on the relationship between flexibility and activity at ambient temperatures.
Highlights
From sequence alignment, Rubiscos can be classified into four groups, types I, II, III, and IV [5]
Type I Rubisco seems to be by far predominant, as it is distributed in all eukaryotes and cyanobacteria as well as in a wide range of other autotrophic bacteria
Type II Rubisco is confined to autotrophic bacteria
Summary
Media, and Growth Conditions—The Tk-Rubisco gene (rbcTk) was isolated from T. kodakarensis KOD1, a hyperthermophilic archaeon isolated from Kodakara Island, Kagoshima, Japan [31, 32]. E. coli DH5␣ was used for gene manipulation and plasmid construction. Gene expression was performed with E. coli BL21(DE3)CodonPlus RIL (Stratagene, La Jolla, CA) for kinetics assays, and with Rosetta2(DE3)pLysS (Novagen, Darmstadt, Germany) for crystallizations. E. coli cells were grown aerobically at 37 °C in Luria-Bertani (LB) medium with ampicillin (100 g/ml for DH5␣ and BL21(DE3)CodonPlus RIL) or carbenicillin (50 g/ml for Rosetta2(DE3)pLysS). R. palustris cells were first cultivated aerobically in the dark at 30 °C in LB medium containing 0.3% NaCl and zeocin (400 g/ml) (heterotrophic growth) for 2 days. Cells were inoculated at an initial A660 of 0.05 into BSA medium, and photoheterotrophically incubated with a light intensity of 3,200 lux at 25 °C. Cells were grown in a sealed glass vial filled with medium so that the head space was minimal.
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