The primary assimilation of carbon dioxide into organic matter is catalyzed by the enzyme ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO). This enzyme also has the capacity to act as an internal monooxygenase and catalyzes the oxygenolytic cleavage of RuBP under suitable conditions. RubisCO is thus a bifunctional protein in which the same polypeptide chain catalyzes the fist step of two competing reactions of cellular metabolism; RubisCO is also the most abundant protein found on earth. This study is directed at two major areas of protein structure and function: the mechanism of complex oligomer assembly and the role of diverse domains in RubisCO catalysis. Recombinant large and small subunits of RubisCO have been purified by separate expression of the structural genes in Escherichia coli and the complex active oligomeric protein has been reconstituted in vitro. In addition, certain residues of the small subunit that affect assembly and influence catalysis have been identified by mutation. Thus, a major goal of the proposed work will focus on the interaction of various subunits and the role certain residues contribute to assembling the oligomer. The role of chaperonin proteins in mediating the proper folding and subsequent assembly of aggregates of large an small subunits will be probed and we will seek to establish the precise pathway of RubisCO oligomer formation. The second major thrust of our research will consider the influence of small subunits on catalysis by large subunits. Again, aided by recombinant DNA procedures, we have developed a convenient system to research this goal. A number of mutant enzymes have been isolated, and will be isolated in the future, to enable us to probe subunit interactions that influence catalysis. The specific domains of the large subunit which contribute to discrimination between the gaseous substrates will also be probed by a combination of molecular manipulation of specific sequences and random selection of strains that express RubisCO with altered properties. Finally, in vivo posttranslational regulation of RubisCO activity will be studied in bacteria and the molecular basis of an apparent reversible modification of RubisCO will be investigated. These studies thus present an excellent opportunity to relate control of cellular metabolism to the function and assembly of an important, yet complex, oligomeric protein.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM024497-18
Application #
2174284
Study Section
Biochemistry Study Section (BIO)
Project Start
1989-01-01
Project End
1996-07-31
Budget Start
1995-01-01
Budget End
1996-07-31
Support Year
18
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Ohio State University
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
098987217
City
Columbus
State
OH
Country
United States
Zip Code
43210
Singh, Jaya; Tabita, F Robert (2010) Roles of RubisCO and the RubisCO-like protein in 5-methylthioadenosine metabolism in the Nonsulfur purple bacterium Rhodospirillum rubrum. J Bacteriol 192:1324-31
Satagopan, Sriram; Scott, Stephanie S; Smith, Todd G et al. (2009) A Rubisco mutant that confers growth under a normally ""inhibitory"" oxygen concentration. Biochemistry 48:9076-83
Imker, Heidi J; Singh, Jaya; Warlick, Benjamin P et al. (2008) Mechanistic diversity in the RuBisCO superfamily: a novel isomerization reaction catalyzed by the RuBisCO-like protein from Rhodospirillum rubrum. Biochemistry 47:11171-3
Tabita, F Robert; Hanson, Thomas E; Satagopan, Sriram et al. (2008) Phylogenetic and evolutionary relationships of RubisCO and the RubisCO-like proteins and the functional lessons provided by diverse molecular forms. Philos Trans R Soc Lond B Biol Sci 363:2629-40
Tabita, F Robert; Satagopan, Sriram; Hanson, Thomas E et al. (2008) Distinct form I, II, III, and IV Rubisco proteins from the three kingdoms of life provide clues about Rubisco evolution and structure/function relationships. J Exp Bot 59:1515-24
Tabita, F Robert; Hanson, Thomas E; Li, Huiying et al. (2007) Function, structure, and evolution of the RubisCO-like proteins and their RubisCO homologs. Microbiol Mol Biol Rev 71:576-99
Kreel, Nathaniel E; Tabita, F Robert (2007) Substitutions at methionine 295 of Archaeoglobus fulgidus ribulose-1,5-bisphosphate carboxylase/oxygenase affect oxygen binding and CO2/O2 specificity. J Biol Chem 282:1341-51
Li, Huiying; Sawaya, Michael R; Tabita, F Robert et al. (2005) Crystal structure of a RuBisCO-like protein from the green sulfur bacterium Chlorobium tepidum. Structure 13:779-89
Finn, Michael W; Tabita, F Robert (2004) Modified pathway to synthesize ribulose 1,5-bisphosphate in methanogenic archaea. J Bacteriol 186:6360-6
Smith, Stephanie A; Tabita, F Robert (2004) Glycine 176 affects catalytic properties and stability of the Synechococcus sp. strain PCC6301 ribulose-1,5-bisphosphate carboxylase/oxygenase. J Biol Chem 279:25632-7

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