title: An evolutionally conserved Lys122 is essential for function in Rhodospirillum rubrum bona fide RuBisCO and Bacillus subtilis RuBisCO-like protein.
abstract: Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and RuBisCO-like protein (RLP) catalyze similar enolase-type reactions. Both enzymes have a conserved non-catalytic Lys122 or Arg122 on the beta-strand E lying in the interface between the N- and C-terminal domains. We used site-directed mutagenesis to analyze the function of Lys122 in the form II Rhodospirillum rubrum RuBisCO (RrRuBisCO) and Bacillus subtilis RLP (BsRLP). The K122R mutant of RrRuBisCO had a 40% decrease in k(cat) for carboxylase activity, a 2-fold increase in K(m) for CO2, and a 1.9-fold increase in K(m) for ribulose-1,5-bisphosphate. K122M and K122E mutants of RrRuBisCO were almost inactive. None of the substitutions affected the thermal stability of RrRuBisCO. The K122R mutant of BsRLP had a 32% decrease in k(cat) and lower thermal stability than the wild-type enzyme. The K122M and K122E mutants of BsRLP failed to form a catalytic dimer. Our results suggest that the lysine residue is essential for function in both enzymes, although in each case, its role is likely distinct.
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), 8916-5 Takayama, Ikoma, Nara 630-0101, Japan.
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Nakano
authors.forename: Hiroki
authors.initials: H
authors.lastname: Ashida
authors.forename: Eiichi
authors.initials: E
authors.lastname: Mizohata
authors.forename: Hiroyoshi
authors.initials: H
authors.lastname: Matsumura
authors.forename: Akiho
authors.initials: A
authors.lastname: Yokota
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D017124:Conserved Sequence; D004795:Enzyme Stability; D019143:Evolution, Molecular; D006358:Hot Temperature; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D055503:Protein Multimerization; D012247:Rhodospirillum rubrum / Q000201:enzymology* / Q000235:genetics; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012274:Ribulosephosphates / Q000378:metabolism
chemical_list: D012274:Ribulosephosphates; C001933:ribulose-1,5 diphosphate; D012273:Ribulose-Bisphosphate Carboxylase; D008239:Lysine