Dissertation/Thesis Abstract

Examination of mutants that alter oxygen sensitivity and co2/o2 substrate specificity of the ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) from archaeoglobus fulgidus
by Kreel, Nathaniel Edward, Ph.D., The Ohio State University, 2008, 168; 10631118
Abstract (Summary)

The archaeon Archaeoglobus fulgidus contains a gene (rbcL2) that encodes the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the enzyme necessary for biological reduction and assimilation of CO2 to organic carbon. Based on sequence homologies and phylogenetic differences, archaeal Rubiscos represent a special class of Rubisco, termed form III, that distinguishes it from the previously characterized form I and form II enzymes. Form III Rubisco retains many features characteristic of all forms of Rubisco, yet exhibits many interesting and unique differences that might be exploited to learn more about structure-function relationships for this protein. For example, recombinant A. fulgidu RbcL2 was shown to possess an extremely high kcatvalue (23 s1) and optimal activity was reached at temperatures up to 93°C. Furthermore, this protein was unusual in that exposure or assay in the presence of O2 (in the presence of high levels of CO 2) resulted in substantial loss (90%) in activity compared to assays performed under strictly anaerobic conditions. Kinetic studies indicated that A. fulgidus RbcL2 possessed an unusually high affinity for O 2. Comparative bioinformatic analyses of available archaeal Rubisco sequences suggested the potential importance of several unique residues, as did further analyses within the context of available forms I/II/III Rubisco structures. Two residues unique to archaeal Rubisco, Met-295 and Ser-363, were of particular interest due to their proximity to known active site residues. Moreover, it was shown that recombinant mutant M295D, S363I and S363V A. fulgidu enzymes were less sensitive to oxygen compared to the wild-type protein. The unique oxygen sensitivity of this form III archaeal Rubisco is being used as a model to provide clues as to how Rubisco has evolved to become more stable in the presence of oxygen in more evolutionarily advanced form I and form II proteins. In addition, the same mutational changes were made at identical sites in Thermococcus kodakaraensis RbcL which shares a 72% amino acid sequence identity with A. fulgidu RbcL2. Although this Rubisco enzyme from T. kodakaraensis is not as oxygen sensitive as the Rubisco from A. fulgidu, there are many similarities in oxygen sensitivity not observed in other form I and form II Rubiscos. A. fulgidu RbcL2 is able to complement growth in the double Rubisco knockout strain SBI/II- from Rhodobacter capsulatus. For the first time, using the A. fulgidu RbcL2 enzyme to complement growth in this knockout strain has enabled the wild-type and mutant enzymes to be studied in vivo.

Indexing (document details)
Advisor: Tabita, F. Robert
Commitee:
School: The Ohio State University
Department: Ohio State Biochemistry Program
School Location: United States -- Ohio
Source: DAI-B 78/11(E), Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Biochemistry
Keywords: Archaeoglobus fulgidus, Biochemistry, Mutagenesis, Rubisco
Publication Number: 10631118
ISBN: 978-0-355-01413-6
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