Structural Perturbations of Rhodopseudomonas palustris Form II RuBisCO Mutant Enzymes That Affect CO₂ Fixation

Abstract

The enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and its central role in capturing atmospheric CO 2 via the Calvin−Benson−Bassham (CBB) cycle have been well-studied. Previously, a form II RuBisCO from Rhodopseudomonas palustris, a facultative anaerobic bacterium, was shown to assemble into a hexameric holoenzyme. Unlike previous studies with form II RuBisCO, the R. palustris enzyme could be crystallized in the presence of the transition state analogue 2-carboxyarabinitol 1,5bisphosphate (CABP), greatly facilitating the structure−function studies reported here. Structural analysis of mutant enzymes with substitutions in form II-specific residues (Ile165 and Met331) and other conserved and semiconserved residues near the enzyme's active site identified subtle structural interactions that may account for functional differences between divergent RuBisCO enzymes. In addition, using a distantly related aerobic bacterial host, further selection of a suppressor mutant enzyme that overcomes negative enzymatic functions was accomplished. Structure−function analyses with negative and suppressor mutant RuBisCOs highlighted the importance of interactions involving different parts of the enzyme's quaternary structure that influenced partial reactions that constitute RuBisCO's carboxylation mechanism. In particular, structural perturbations in an intersubunit interface appear to affect CO 2 addition but not the previous step in the enzymatic mechanism, i.e., the enolization of substrate ribulose 1,5-bisphosphate (RuBP). This was further substantiated by the ability of a subset of carboxylation negative mutants to support a previously described sulfur-salvage function, one that appears to rely solely on the enzyme's ability to catalyze the enolization of a substrate analogous to RuBP.

An Insight of RuBisCO Evolution through a Multilevel Approach

“…Moreover, the work of Satagopan et al [63] on R. rubrum (RuBisCO isoform II) is in agreement with Watanabe et al [79], because the link between the αB and βC loop with loop 6 was also identified; this link allows the conformation of the catalytic loop to be stabilized. Satagopan et al [63] used mutants (5HAN S59F and 5HJX A47V ) in R. rubrum where CO 2 was the only carbon source; their results showed a similar biological growth between mutants and a decrease with respect to WT [63,80]. Thus, our PCA results of DM indicated that mutants (5HAN S59F and 5HJX A47V ) would have undergone a change from an unstable intermediate conformation (4LF1 WT ) to an unstable one, showing a greater variation in the conformational distribution of the RuBisCO (Figure 7b,c), being consistent with the RMSD results where 5HJX A47V was more flexible (~0.1 nm) than WT (Figure 5).…”

“…In Figure 4, the eNMA shows the consensus fluctuations are highlighted and reveal a conserved pattern among species and RuBisCO forms (Figure 4a,b). The three isoforms show a greater fluctuation in the N-terminal domain spanning the amino acid residues (51)(52)(53)(54)(55)(56)(57)(58)(59)(60)(61)(62)(63)(64)(65)(66)(67)(68) between the secondary elements αB and βC (Figure 4a), which are functionally relevant for RbcL. Likewise, RuBisCO form III presents greater fluctuation (≥3 Å) with respect to form I and II (Figure 4a,b).…”

“…Changes were found at loop 6 and in the loop between αB and βC, which is a critical region for gaseous substrate binding after RuBP enolization has been completed [18,63]. Moreover, the comparison between 4LF1 WT and 5HJX A47V allowed the identification of key residues that increased (Gly35, Lys330−Met331) and reduced (Val201−Phe202) RuBisCO form II fluctuation by more than 0.1 nm (Table 1), which can be attributed to A47V mutation and which has an effect on RuBisCO catalytic activity [63]. The RMSD analysis of form III showed that the 3A12 WT protein (~0.18 nm ± 0.001) was more stable than 3KDO SP6 (~0.28 nm ± 0.001) and 3WQP T289D (~0.22 nm ± 0.001); see Table 1.…”

Insights into the Structure of Rubisco from Dinoflagellates-In Silico Studies

“…Such conclusion was drawn based on their homology to the R. rubrum enzyme, which functions exclusively as a dimer. However, our study indicated that there is a high homology of dinoflagellate Rubisco to the same enzyme of R. palustris, shown recently to be a hexamer [30]. An indication of the possibility of a hexamer formation by Symbiodinium Rubisco may come from an analysis of the probable oligomerization interface.…”

Inside‐and‐Out Semiconductor Engineering for CO2 Photoreduction: From Recent Advances to New Trends

“…Photosynthetic organisms develop into metabolic pathways to realize CO 2 reduction and store solar energy in chemicals. [ 413,414 ] Biologic carbon fixation has made great achievements that artificial catalysts are not able to make, such as the lifetime of enzymes and specificity of catalytic pathways, [ 415–421 ] But the light absorption capability of semiconductors is often higher than that of natural photosynthesis. [ 422–425 ] Thus, it is a promising way to solve CO 2 reduction issues by integrating high light‐absorbing inorganic catalysts with photosynthetic organisms.…”