title: 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.
authors.affiliation: Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Martinsried, Germany.
authors.forename: Paulo
authors.initials: P
authors.lastname: Durão
authors.affiliation: Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Martinsried, Germany.
authors.forename: Harald
authors.initials: H
authors.lastname: Aigner
authors.affiliation: Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Martinsried, Germany.
authors.forename: Péter
authors.initials: P
authors.lastname: Nagy
authors.affiliation: Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Martinsried, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Mueller-Cajar
authors.affiliation: Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Martinsried, Germany.
authors.forename: F Ulrich
authors.initials: FU
authors.lastname: Hartl
authors.affiliation: Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Martinsried, Germany.
authors.forename: Manajit
authors.initials: M
authors.lastname: Hayer-Hartl
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019020:Directed Molecular Evolution / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010788:Photosynthesis; D017510:Protein Folding; D055503:Protein Multimerization; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046940:Synechococcus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D046939:Synechocystis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C028715:GroE protein, E coli; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; C522129:RbcX protein, cyanobacteria; D012273:Ribulose-Bisphosphate Carboxylase