Description

Broadly, this project addresses the biological control mechanisms that limit the conversion of energy derived from photosynthetic electron transport into chemical storage forms such as carbohydrates. Net photosynthesis on earth is limited by the slow turnover rate of the enzyme Rubisco, which restricts the rate of atmospheric carbon incorporation into biological systems and therefore constitutes the bottleneck of photosynthesis. RuBisCo, the most abundant protein on earth, catalyzes the sequestration of carbon dioxide from the atmosphere and its incorporation into simple carbohydrates that are fed into the cellular metabolism. In higher plants and green algae, the auxiliary protein Rubisco activase plays an essential role in maintaining RuBisCo in its active state, and is therefore indispensable for these organisms to stay alive and thrive. The activase is thought to play an essential role in a complex set of regulatory mechanisms that limit net CO2 assimilation at temperatures above the thermal optimum. As the elevation of greenhouse gases in the earths atmosphere appears to be coupled to rising global temperatures, efforts aimed at increasing the net rate of biological carbon fixation under heat conditions will continue to occupy a central theme in bioenergy. This project is focused onto utilizing biophysical and biochemical methods to study structural and mechanistic features of Rubisco activase, and its mode of interaction with Rubisco. The activase is thought to function as a molecular motor, responsible for removing tight-binding inactivators that rapidly accumulate in the catalytic sites of Rubisco. To address the heat lability of Rubisco activation, a detailed understanding of the mechanism of Rubisco regulation by its activase is essential. Long-term, the research proposed here is expected to have significant impact on the development of reliable mathematical models useful in the prediction of net biomass production by land plants and green algae under moderate heat stress conditions. .z

Description

This project addresses the regulation of higher plant carbon assimilation by Rubisco activase (Rca), a chemo-mechanical motor protein essential in maintaining Rubisco activity. Rca is a critical component of a complex set of biological feed-back loops that regulate the conversion of energy derived from photosynthetic electron transport into chemical storage forms such as carbohydrates. The goal of this work is to utilize biophysical and spectroscopic methods to elucidate atomic-resolution structures of Rca, to shed light on the dynamic assembly-disassembly processes responsible for maintaining Rubisco activity, and to investigate the mode of interaction of Rca with its partner protein Rubisco. Long-term, we are interested in gaining a full understanding of the mechanism of Rubisco remodeling by Rca from a structural and mechanistic point of view. The extremely abundant enzyme Rubisco catalyzes the incorporation of atmospheric CO2 into simple carbohydrates, thus generating the necessary precursors for hexose biosynthesis. In many photosynthetic organisms, the auxiliary protein Rca has been recruited to catalyze the rapid release of trapped inhibitors from Rubisco sites to reactivate Rubisco for CO2 fixation. Rca belongs to a group of P-loop ATPases that operate in ring-like assemblies to conformationally remodel their partner proteins. In turn, Rca is heavily regulated by the ambient ATP/ADP ratio in the chloroplast stroma, and in longer isoforms, by the stromal redox poise. Therefore, Rca constitutes an important regulatory checkpoint responsible for maintaining Rubisco turnover under stromal conditions of high energy charge, and for down-regulating carbon fixation under conditions of low energy charge or heat stress. In a broader sense, we aim to understand how the energy charge of the chloroplast stroma is utilized to regulate biological carbon fixation.

Description

This project addresses the regulation of higher plant carbon assimilation by Rubisco activase (Rca), a chemo-mechanical motor protein essential in maintaining Rubisco activity. Rubisco is the primary carbon fixing enzyme in nature. Its partner protein Rca is a ring-forming ATPase that plays a critical role in regulating the conversion of energy derived from photosynthetic electron transport into chemical storage forms. The proposed work aims to employ biophysical, X-ray crystallographic, spectroscopic and mechanistic approaches to answer fundamental questions in Rubisco regulation by Rca. This project seeks to determine atomic resolution structures of Rca conformational states that are part of the catalytic cycle, to delineate the thermodynamic properties of the physical interaction between Rca and Rubisco, and to unravel the mechanistic enzymology of ATPase and Rubisco reactivation activities by use of kinetic methods. Long-term, we are interested in gaining a comprehensive understanding of the mechanism of Rubisco remodeling from a structural and dynamic point of view.
Higher plant Rubisco requires the synergistic action of several enzymes to cope with self-inhibition intrinsic to turnover in an oxygenic atmosphere. With decreasing CO2 and increasing O2, increasing amounts of xylulose bisphosphate (XuBP) and pentodiulose bisphosphate (PDBP) are produced, compounds that resemble the substrate and prevent Rubisco activity by obstructing its active sites. For Rubisco to resume CO2 assimilation, these compounds must first escape from the active site, a process substantially accelerated by the motor protein Rca. In addition, Rca plays a key role in the release of ribulose bisphosphate (RuBP) from decarbamylated Rubisco and in the release of the nocturnal inhibitor carboxyarabinitol phosphate (CA1P) that is produced in the stroma of some plants.
Arguably, the ultimate goal is to develop the predictive power required for the rational engineering of metabolic pathways for improved biomass accumulation. In spite of the enormous complexity surrounding Rubisco function, the work proposed here aims to provide a significant milestone towards a comprehensive understanding of all co-regulatory mechanisms that enhance or diminish the rate of biological carbon fixation in higher plants.

Description

This project is focused on the regulation of higher plant carbon assimilation by Rubisco activase (Rca), a chemo-mechanical motor protein essential in maintaining ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity. Rca is responsible for accelerating the release of tight-binding inhibitors from Rubisco active sites, and therefore plays a key role in regulating the conversion of energy derived from photosynthetic electron transport into chemical storage forms. Rca activity has been shown to be critical for continued carbon fixation under high light and for the regulation of dark-light transitions. Rca is a ring-forming P-loop ATPase that belongs to the superfamily of AAA-plus proteins known for their macromolecular remodeling functions.
The overall goal of this project is to elucidate the relationship between Rca structure, ATPase activity and Rubisco reactivation activity. The first aim is to use mechanistic enzymology to understand the roles of post-translational modifications and divalent cations in Rca regulation. The second aim is to use single-molecule work to understand self-association, nucleotide binding and site asymmetry, and the third aim is to characterize the structure free and Rubisco-bound Rca.
StatusActive
Effective start/end date9/15/099/14/20

Funding

  • US Department of Energy (DOE): $2,535,000.00

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Calvin cycle
heat inactivation
ribulose 1,5-diphosphate
oxygenases
Chlorophyta
assimilation (physiology)
carbon dioxide
energy conversion
adenosinetriphosphatase
proteins
active sites
electron transfer
ribulose-bisphosphate carboxylase activase
enzymology
carbohydrates
ribulose-bisphosphate carboxylase
enzymes