Download Session 1B - Rubisco and Photorespiration

Session 1B - Rubisco and Photorespiration
The interplay between photorespiration, the Calvin-Benson cycle and other metabolism
Hermann Bauwe
Department of Plant Physiology, University of Rostock, D-18051 Rostock, Germany
The Calvin-Benson cycle and the photorespiratory pathway form the photosynthetic-photorespiratory
supercycle that is responsible for nearly all biological CO2 fixation on Earth. In essence, supplementation
with the photorespiratory pathway is necessary because ribulose 1,5-bisphosphate carboxylase, the CO2fixing enzyme of the Calvin-Benson cycle, catalyses several side reactions including the oxygenation of
ribulose 1,5-bisphosphate, which produces the noxious metabolite phosphoglycolate. The
photorespiratory pathway recycles the phosphoglycolate to 3-phosphoglycerate and in this way allows the
Calvin-Benson cycle to operate in the presence of molecular oxygen. However, the photorespiratory
pathway contributes to cellular metabolism on a much broader scale. This talk will discuss regulatory
feedback from photorespiration to chloroplastidal metabolism such as the Calvin-Benson cycle and starch
synthesis, the role of photorespiration as an inter-organelle transportation highway for reducing
equivalents that drives ATP production in the mitochondrion and subsequently sucrose synthesis in the
cytosol, and new data on the linking up of photorespiration with nitrogen metabolism.
Acknowledgement: German ScienceFoundation, Research Unit FOR 1186 (Promics) Improving plant photosynthesis and growth via Rubisco engineering
Spencer Whitney, The Australian National University. Acton, Australia
The photosynthetic enzyme linking the inorganic and organic phases of the biosphere is Ribulose-1,5bisphosphate [RuBP] carboxylase/oxygenase (Rubisco). Encumbering the CO2-fixing performance of all
Rubisco isoforms is a complicated catalytic chemistry that slows turnover rate (kcat ~1 to 13 s-1), allows for
competitive inhibition by oxygen and can produce misfire products that can self-inhibit activity. Significant
effort has been invested into better understanding the structure-function details of Rubisco as improving
its performance is recognised as a viable means to enhance the photosynthetic efficiency and yield
potential of crops. Simulations of natural kinetic diversity has identified Rubisco variants of potential
benefit to C3-photosynthesis with sequence analyses highlighting amino acids under evolutionary
selection pressure with theoretical predictions used to speculate those that might influence catalysis. The
challenge is taking the next step to translate simulation into reality. In this talk I will provide examples of
new technologies and their limitations to bioengineering different Rubisco phylogenies in leaf
chloroplasts. Shown will be a demonstration of the successful use of directed evolution and ancillary
protein co-engineering to improve Rubisco performance that translate to supporting higher rates of leaf
photosynthesis and improved plant growth.
A unique Large-subunit loop acts as an in-built Small-subunit mimic by concentrating
large subunits in Rubisco from Methanococcoides burtonii.
Laura Gunn, Karin Valegård, Inger Andersson
Uppsala University, Uppsala, Sweden
The CO2-fixing enzyme Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is comprised of
dimers of catalytic Large subunits (LSu, ≈50 kDa). Form I Rubiscos also contain Small subunits (SSu, ≈ 15
kDa), which glue Lsu dimers (L2) together. The Rubisco isoform from the methanogenic archaeon
Methanococcoides burtonii is comprised solely of LSu dimers; five M. burtonii Rubisco (MbR) L2 dimers
oligomerise to form a (L2)5 enzyme in a substrate-dependent manner. The class of Rubiscos to which MbR
belongs is not clear. Despite M. burtonii’s close evolutionary relationship to form III Rubisco-containing
species and MbR exhibiting form III-like decameric assembly, MbR exhibits higher sequence homology to
form II Rubiscos. Furthermore, MbR contains an entirely unique 30-amino-acid insertion or “bonus
sequence” near the C-terminus that is essential for oligomerisation. Despite MbR’s unique phylogeny and
sequence, it remains structurally uncharacterised and the role of the bonus sequence in oligomerisation
remains obscure. In this study, MbR was heterologously expressed in E. coli, the MbR structure probed by
X-ray crystallography, and the importance of specific residues for MbR oligomerisation confirmed using sitespecific mutagenesis. Here we present the first 3D X-ray crystal structure of (L2)5 MbR clearly showing the
unique structure of the bonus sequence that acts as an in-built SSu, tethering together MbR L2 dimers. The
identification of this novel LSu-concentrating feature combined with analyses of LSu phylogeny and
distribution of the bonus sequence suggest that MbR represents a new subclass of Rubisco isoforms.
Assembly and Regulation of Higher Plant Rubisco Activase
Rebekka Wachter1, Marcia Levitus1, W. E. Moerner2, Dayna S. Peterson1, Matthew T. Hilton1, Andrew J. Serban1, J.
Nathan Henderson1, Suratna Hazra1, Quan Wang2
1
Arizona State University, Tempe, United States of America, 2Stanford University, Stanford, United States of America
Higher plant carbon assimilation is regulated by Rubisco activase (Rca), an AAA+ ATPase essential in
maintaining Rubisco activity. To cope with competitive inhibitors, Rubisco must interact with Rca assemblies
capable of coupling ATP hydrolysis to the restructuring of Rubisco sites. Here, we seek to elucidate the
catalytic and regulatory roles played by different oligomeric Rca species. Mechanistic work indicates that
allosteric regulation of nucleotide hydrolysis requires small amounts of ADP. Cooperative magnesium
binding increases the catalytic efficiency by an order of magnitude, suggesting in vivo regulation by lightdependent magnesium fluctuations. To analyze the observed size polydispersity, we are monitoring the
step-wise assembly process of cotton, tobacco and spinach Rca by fluorescence correlation spectroscopy.
We find that the microscopic binding constants differ significantly between species. ATPase activity appears
to spike when the hexamer is dominant, but declines when higher-order states become populated. These
observations suggest that large aggregates could provide a storage mechanism for inactive Rca in the dark.
In related work, we are utilizing the recently developed capability of the ABEL trap to analyse oligomer
distributions at the single-molecule level. Recently, we have developed a FRET-based subunit exchange
assay that allows us to monitor the distribution of fluorescently labeled subunits among different particles.
We find that in some variants, protomer mixing occurs solely during periods of rapid hydrolytic activity,
whereas ADP and nucleotide analogs inhibit exchange completely. These observations suggest that
Rubisco activation may be tightly linked to the dynamic reorganization of Rca subunits in a highly transient
Rubisco-Rca complex.
Insights into Rubisco activase mechanism and thermostability gleaned from
bioprospecting
Devendra Shivhare, Oliver Mueller-Cajar
Nanyang Technological University, Singapore, Singapore
Ribulose 1, 5-bisphosphate carboxylase/oxygenase (Rubisco) forms inhibited complexes with its own
substrate Ribulose 1, 5-bisphosphate (RuBP) and other sugar phosphates. The AAA+ protein Rubisco
activase (Rca) counteracts this issue by removing these inhibitors and thus maintaining Rubisco in its active
state. Inactivation of Rca at moderately high temperatures is associated with the loss of the activation state
of Rubisco. Here in a comparative study with rice (Oryza sativa), we describe a highly functional and
thermostable Rca (tsRca) system from another monocot species. Recombinantly produced tsRca isoforms
were found to exhibit ~4-fold increased rice Rubisco activation rates in vitro compared to the cognate OsRca
at 25 °C. tsRca maintained significant ATPase hydrolysis activity at 50 °C whereas most OsRca functionality
was lost at 42 °C. Although OsRca and tsRca readily formed hetero-oligomeric complexes, activase
functionality was not perturbed. Upon heating the hetero-oligomer, tsRca subunits remained in solution
while OsRca formed inactive higher order aggregates. Domain swapping experiments localized
thermostability to the nucleotide binding domain of tsRca. No single residue substitutions could be identified
to be responsible for thermostability. However, a single glutamic acid to glutamine (E-Q) substitution
localized at a newly identified surface loop of the activase conferred tsRca’s high activation rate to OsRca.
Further mutational analysis implicated this loop in Rubisco-Rca protein-protein interactions. Our results
indicate that further bioprospecting and careful biochemical characterization of Rca homologues will lead to
both mechanistic insights and identification of candidate proteins for engineering enhanced thermotolerance
in rice.