Opposing effects of folding and assembly chaperones on evolvability of Rubisco

Abstract

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the fixation of CO2 in photosynthesis. Despite its pivotal role, Rubisco is an inefficient enzyme and thus is a key target for directed evolution. Rubisco biogenesis depends on auxiliary factors, including the GroEL/ES-type chaperonin for folding and the chaperone RbcX for assembly. Here we performed directed evolution of cyanobacterial form I Rubisco using a Rubisco-dependent Escherichia coli strain. Overexpression of GroEL/ES enhanced Rubisco solubility and tended to expand the range of permissible mutations. In contrast, the specific assembly chaperone RbcX had a negative effect on evolvability by preventing a subset of mutants from forming holoenzyme. Mutation F140I in the large Rubisco subunit, isolated in the absence of RbcX, increased carboxylation efficiency approximately threefold without reducing CO2 specificity. The F140I mutant resulted in a ∼55% improved photosynthesis rate in Synechocystis PCC6803. The requirement of specific biogenesis factors downstream of chaperonin may have retarded the natural evolution of Rubisco.

Evolutionary trend toward kinetic stability in the folding trajectory of RNases H

“…Whereas the extant homologs have an ∼10-fold slower folding rate than their common ancestor, they still fold efficiently and reversibly, reaching their native folded state within seconds. It is possible that changes in the folding rate may well be a result of neutral drift, and that there may be no selective pressure to optimize folding rates beyond a biologically necessary range (8,41). Modern proteins can be computationally redesigned to fold faster, demonstrating that their folding rates have not been maximized over evolution (42).…”

“…Thus, the energy landscape defines a protein's ability to fold in a biologically reasonable time scale, avoid misfolding, and prevent frequent unfolding-all of which are likely important for the overall fitness of an organism. Indeed, numerous studies have proposed how protein folding might affect molecular evolution (2)(3)(4)(5)(6)(7)(8), and there is growing evidence that folding pathways and their associated kinetics and energetics are evolutionarily constrained. To date, however, there is little experimental evidence detailing how folding properties have changed throughout evolution.…”

Engineering of chimeric eukaryotic/bacterial Rubisco large subunits in <i>Escherichia coli</i>

“…It is likely that some of these changes had no role in post-translational processing, folding or assembly events, but instead effected a global disturbance to protein tertiary structure. For the Section 4 mutant, which has three substitutions, because substituted residues Leu-161 and Met-169 (numbering based on Chlamydomonas RbcL) can be mutated without affecting function (Durão et al, 2015), the remaining P168G (Chlamydomonas RbcL numbering) mutation may be responsible for destabilizing the large subunit fold as a result of the loss of the bulkier and more rigid proline pyrrole group. As for the Section 7 mutant, which has six substitutions, four of the substituted residues on the catalytic Loop 6 were previously changed to spinach-specific residues, but an assembled holoenzyme with decreased activity was still produced .…”

A warm welcome for alternative CO2 fixation pathways in microbial biotechnology

“…Parikh et al ., 2006; Durão et al ., 2015), introducing more efficient photorespiration pathways (e.g. Shih et al ., 2014) and introducing CO 2 concentrating mechanisms to increase the carboxylating activity of RubisCO (e.g.…”