Role of the Rubisco Small Subunit

Technical Report · Sat Nov 05 04:00:00 EDT 2016

DOI:https://doi.org/10.2172/1330984· OSTI ID:1330984

Spreitzer, Robert Joseph ^[1]

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1. Univ. of Nebraska, Lincoln, NE (United States); University of Nebraska-Lincoln

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of CO₂ fixation in photosynthesis. However, it is a slow enzyme, and O₂ competes with CO₂ at the active site. Oxygenation initiates the photorespiratory pathway, which also results in the loss of CO₂. If carboxylation could be increased or oxygenation decreased, an increase in net CO₂ fixation would be realized. Because Rubisco provides the primary means by which carbon enters all life on earth, there is much interest in engineering Rubisco to increase the production of food and renewable energy. Rubisco is located in the chloroplasts of plants, and it is comprised of two subunits. Much is known about the chloroplast-gene-encoded large subunit (rbcL gene), which contains the active site, but much less is known about the role of the nuclear-gene-encoded small subunit in Rubisco function (rbcS gene). Both subunits are coded by multiple genes in plants, which makes genetic engineering difficult. In the eukaryotic, green alga Chlamydomonas reinhardtii, it has been possible to eliminate all the Rubisco genes. These Rubisco-less mutants can be maintained by providing acetate as an alternative carbon source. In this project, focus has been placed on determining whether the small subunit might be a better genetic-engineering target for improving Rubisco. Analysis of a variable-loop structure (βA-βB loop) of the small subunit by genetic selection, directed mutagenesis, and construction of chimeras has shown that the small subunit can influence CO₂/O₂ specificity. X-ray crystal structures of engineered chimeric-loop enzymes have indicated that additional residues and regions of the small subunit may also contribute to Rubisco function. Structural dynamics of the small-subunit carboxyl terminus was also investigated. Alanine-scanning mutagenesis of the most-conserved small-subunit residues has identified a possible structural pathway between the small-subunit βA-βB loop and alpha-helix 8 of the large-subunit α/β-barrel active site. Hybrid enzymes were also created comprised of plant small subunits and Chlamydomonas large subunits, and these enzymes have increases in CO₂/O₂ specificity, further indicating that small subunits may be the key for ultimately engineering an improved Rubisco enzyme.

• Research Organization: Univ. of Nebraska, Lincoln, NE (United States)
• Sponsoring Organization: USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
• DOE Contract Number: FG02-00ER15044
• OSTI ID: 1330984
• Report Number(s): DOE-UNL--ER15044
• Country of Publication: United States
• Language: English

Related Subjects

09 BIOMASS FUELS
14 SOLAR ENERGY
59 BASIC BIOLOGICAL SCIENCES
Biomass
Carbon dioxide fixation
Chlamydomonas
Chloroplast
Enzyme catalysis
Genetic engineering
Photosynthesis
Protein engineering
Protein structure
Ribulosebisphosphate carboxylase/oxygenase
Rubisco