Structure-based Catalytic Optimization of a Type III Rubisco from a Hyperthermophile

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 ␣-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 ␣-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 ␣-helix 6 induced by four essential factors. We thus supposed that an increase in the flexibility of the ␣-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.The Calvin-Benson-Bassham (CBB) 4 cycle contributes to the fixation of carbon for all plants, algae, and cyanobacteria. The most important enzyme in the CBB cycle is ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco: EC 4.1.1.39) (1, 2). Rubisco is responsible for the single carbon dioxidefixing step of the cycle, in which ribulose-1,5-bisphosphate reacts with CO 2 and H 2 O to form two molecules of 3-phosphoglycerate. However, it also catalyzes a competitive oxygenase reaction, by which ribulose-1,5-bisphosphate reacts with O 2 to form one molecule of 3-phosphoglycerate and one molecule of 2-phosphoglycolate (3, 4). Moreover, a typical Rubisco is able to fix only three carbon dioxide molecules/s, an extremely low turnover rate for an enzyme (3). Rubisco is thus considered to be the rate-limiting enzyme of the CBB pathway, and has been an important target for protein engineering (3).From sequence alignment, Rubiscos can be classified into four groups, types I, II, III, and IV (5). The classical type I and II Rubiscos function in the CBB cycle. Type I Rubisco is composed of eight large subunits (L subunits) and eight small subunits (S subunits) with tetragonal symmetry (L 8 S 8 ) (6). Type II Rubisco is usually composed of only two L subunits (L 2 ) (7, 8). In both cases, the L 2 dimer is the catalytic unit of Rubisco, generating two catalytic...

Mutation design of a thermophilic Rubisco based on three‐dimensional structure enhances its activity at ambient temperature

“…Mutant plasmids for gene expression in E. coli were constructed by inverse PCR with pET‐21a(+) harboring the gene of wild‐type Tk ‐Rubisco or SP8 mutant as templates . Primers purified with HPLC were purchased from Hokkaido System Science.…”

“…The best mutant of the second generation, SP5‐V330T (hereafter called SP8; Supporting Information Fig. S1), supports a 55% increase in specific growth rates when introduced into R. palustris Δ3 cells compared to cells introduced with the wild‐type Tk ‐Rubisco …”

“…S1), displayed a 31% increase in specific growth rate compared to that of cells harboring wild‐type Tk ‐Rubisco . Crystal structures of the SP6 mutant and wild‐type Tk ‐Rubisco revealed several factors in the vicinity of helix‐6 that might further enhance carboxylase activity . This information was applied to produce second‐generation mutants.…”

Photosynthetic Microorganisms

“…Interestingly, the phototrophic purple non-sulfur bacteria (e.g., Rhodobacter sphaeroides, Rhodobacter capsulatus) and other organisms including Hydrogenovibrio marinus and some Thiobacillus species contain both form I and form II RuBisCO, and in R. capsulatus both forms are expressed under photoautotrophic conditions (Badger and Bek 2008). Form III is found in archaea and consists of a large subunit in a dimeric or pentameric arrangement (Andersson 2008 (Bracher et al 2011), form II (Tabita et al 2008), form III (Nishitani et al 2010), and form IV (Tabita et al 2007) the RLPs do not seem to be functionally related to RuBisCO, they do, however, share a common sequence identity but do not maintain some key residues present in RuBisCO that are required for catalytic activity. Thus, one may speculate that the lack of these key residues is the reason for RLP's inability to catalyze the RuBisCO CO 2 /O 2 bonafide reaction.…”

Carbohydrate Metabolism in Archaea: Current Insights into Unusual Enzymes and Pathways and Their Regulation

“…All three enzymes have been studied extensively, and the crystal structures from archaeal R1,5BP isomerase and type III RubisCOs have been reported (386)(387)(388)(389)(390)(391)(392)(393)(394)(395)(396). The conversion of nucleoside monophosphates to 3PG was also detected in crude extracts of Tco.…”