title: Residues in three conserved regions of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase are required for quaternary structure.
abstract: To explore the role of individual residues in the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), small subunits with single amino acid substitutions in three regions of relative sequence conservation were produced by directed mutagenesis of the rbcS gene from Anabaena 7120. These altered small subunits were cosynthesized with large subunits (from an expressed Anabaena rbcL gene) in Escherichia coli. Mutants were analyzed for effects on quaternary structure and catalytic activity. Changing Glu-13S (numbering used is that of the spinach enzyme) to Val, Trp-67S to Arg, Pro-73S to His, or Tyr-98S to Asn prevented accumulation of stable holoenzyme. Interpretation of these results using a model for the three-dimensional structure of the spinach enzyme based on x-ray crystallographic data suggests that our small subunit mutants containing substitutions at positions 13S and 67S probably do not assemble because of mispairing or nonpairing of charged residues on the interfacing surfaces of the large and small subunits. The failure of small subunits substituted at positions 73S or 98S to assemble correctly may result from disruption of intersubunit or intrasubunit hydrophobic pockets, respectively.
authors.affiliation: Michigan State University-Department of Energy Plant Research Laboratory, East Lansing 48824.
authors.forename: J H
authors.initials: JH
authors.lastname: Fitchen
authors.forename: S
authors.initials: S
authors.lastname: Knight
authors.forename: I
authors.initials: I
authors.lastname: Andersson
authors.forename: C I
authors.initials: CI
authors.lastname: Branden
authors.forename: L
authors.initials: L
authors.lastname: McIntosh
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
chemical_list: D046911:Macromolecular Substances; D012273:Ribulose-Bisphosphate Carboxylase