Substitutions at methionine 295 of Archaeoglobus fulgidus ribulose-1,5-bisphosphate carboxylase/oxygenase affect oxygen binding and CO2/O2 specificity.
Archaeoglobus fulgidus RbcL2, a form III ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), exhibits unique properties not found in other well studied form I and II Rubiscos, such as optimal activity from 83 to 93 degrees C and an extremely high kcat value (23 s-1). More interestingly, this protein is unusual in that exposure or assay in the presence of oxygen and high levels of CO2 resulted in substantial loss (85-90%) of activity compared with assays performed under strictly anaerobic conditions. Kinetic studies indicated that A. fulgidus RbcL2 possesses an unusually high affinity for oxygen (Ki=5 microM); O2 is a competitive inhibitor with respect to CO2, yet the high affinity for O2 presumably accounts for the inability of high levels of CO2 to prevent inhibition. Comparative bioinformatic analyses of available archaeal Rubisco sequences were conducted to provide clues as to why the RbcL2 protein might possess such a high affinity for oxygen. These analyses suggested the potential importance of several unique residues, as did additional analyses within the context of available form I-III Rubisco structures. One residue unique to archaeal proteins (Met-295) was of particular interest because of its proximity to known active-site residues. Recombinant M295D A. fulgidus Rubisco was less sensitive to oxygen compared with the wild-type enzyme. This residue, along with other potential changes in conserved residues of form III Rubiscos, may provide an understanding as to how Rubisco may have evolved to function in the presence of air.
Kreel
Nathaniel E
NE
Department of Microbiology and the Plant Molecular Biology/Biotechnology Program, Ohio State University, Columbus, Ohio 43210-1292, USA.
Tabita
F Robert
FR
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019611:Archaeoglobus fulgidus / Q000201:enzymology* / Q000235:genetics*; D001665:Binding Sites; D002245:Carbon Dioxide / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity
D002245:Carbon Dioxide; D008715:Methionine; D012273:Ribulose-Bisphosphate Carboxylase; D010100:Oxygen