Rubisco sequence-structure-function : coevolution and codon bias of rbcL gene
Creators: name: Agrawal, Animesh K.
Publisher: name: The Australian National University
Year: 2012
Subjects: subject: QK898.E58 A357 2012
subject: Plant enzymes
subject: Coevolution
subject: Genetics
subject: FOS: Biological sciences

description: Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) is the primary photosynthetic enzyme responsible for C0{u2082} fixation. Though Rubisco is critical to photosynthesis, it is a very inefficient enzyme with a low catalytic rate and competing oxygenase activity that initiates a wasteful photorespiratory pathway. This paradoxical relationship between the functional significance of Rubisco and its apparent inefficiency is puzzling and raises questions regarding the roles of evolution versus functional constraints in shaping Rubisco. This thesis examines the role of coevolution and codon-usage bias of the rbcL gene in Rubisco's fine-tuning at the molecular level. Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) is the primary photosynthetic enzyme responsible for C0{u2082} fixation. Though Rubisco is critical to photosynthesis, it is a very inefficient enzyme with a low catalytic rate and competing oxygenase activity that initiates a wasteful photorespiratory pathway. This paradoxical relationship between the functional significance of Rubisco and its apparent inefficiency is puzzling and raises questions regarding the roles of evolution versus functional constraints in shaping Rubisco. This thesis examines the role of coevolution and codon-usage bias of the rbcL gene in Rubisco's fine-tuning at the molecular level. The extent of information available on Rubisco is substantial. A local database was developed to archive Rubisco protein and nucleotide sequences in public databases, as well as to integrate the structural, kinetic and taxonomic data available on Rubisco. This database is based on BioSQL schema, employs MySQL as the relational database backend and uses the Biopython application programming interface (API). This local repository contains more than 11,000 unique Rubisco large-subunit (LSU) protein/rbcL nucleotide sequence entries from Angiosperms; kinetic data information from 40 species, including 11 species from flowering plants; and structural information from 50 PDB structures, including spinach, tobacco and rice from flowering plants. Coevolution of Rubisco has been investigated in the intra-protein context using a large sequence dataset of Rubisco-LSU sequences, as well as in the inter-protein context through its interactions with Rubisco small subunit, chaperonins RbcX and Rubisco activase (RA). The intra-protein studies identified a novel cluster of coevolving sites spatially proximal to loop 6 and in the C- terminal tail, known regions of functional and structural importance in the Rubisco-LSU. The inter-protein analyses of the Rubisco-LSU and RA detected several new coevolving sites both in the Rubisco-LSU and in RA, in addition to predicting sites already identified by mutagenesis studies to be involved in Rubisco-LSU-RA interaction. In the Rubisco-LSU, these sites are located in the {u03B2}-C-{u03B2}-D loop which is known to be interacting with RA, along with a network of polar/charged residues in the C-terminal domain of the Rubisco-LSU. The codon-usage bias of rbcL was analyzed using a large set of rbcL sequences and all available Angiosperm chloroplast nucleotide sequence data. Consistent with previous reports, studies on Angiopserm chloroplast genomes and their corresponding rbcL genes showed that both the rbcL genes and chloroplast genomes have obvious A+ T bias. Based on evidence found in this study, a role for codon adaptation in rbcL is proposed, alt hough it is limited to several two-fold and a three-fold codon-degenerate amino acids. Significantly, this study show that Rubisco's catalytic residues favor preferred codons (codons used more frequently in rbcL than other synonymous codons). Another important f inding suggests translational accuracy selection in rbcL, based on statistically significant associations of preferred codons in rbcL with conserved and buried sites in the RubiscoLSU. Overall in this thesis, information available on Rubisco has been structured and integrated to develop a high-quality dataset for systematic studies, Rubisco's coevolution has been studied in both intra- and inter-protein contexts to identify coevolutionary constraints in its evolution, and codon preferences of rbcL has been investigated in order to understand the role of synonymous codons within the context of Rubisco's structure/function.