title: Characterization of cysteine residues involved in the reductive activation and the structural stability of rapeseed (Brassica napus) chloroplast fructose-1,6-bisphosphatase.
abstract: In higher plants, light enhances the activity of chloroplast fructose-1,6-bisphosphatase via a cascade of thiol/disulfide exchanges. We have examined the structural and functional role of seven conserved cysteine residues in the rapeseed (Brassica napus) enzyme by site-directed mutagenesis. After lysis of Escherichia coli cells, C53S and C191S variants partitioned mainly in the insoluble fraction whereas C96S, C157S, C174S, C179S, and C307S mutants were soluble. Homogeneous preparations of the latter hydrolyzed fructose 1,6-bisphosphate at similar rates in the presence of 10 mM Mg2+ but only C157S, C174S and C179S mutants were both efficient catalysts at 1 mM Mg2+ and nearly insensitive to dithiothreitol. These results demonstrate the contribution of Cys53 and Cys191 to the stability of the enzyme and the participation of Cys157, Cys174 and Cys179 in the reductive process responsive of the light-dependent regulation. Given that mutations at Cys96 and Cys307 neither destabilize the enzyme nor affect the reductive modulation, their function remains unknown.
authors.affiliation: Instituto de Investigaciones Bioquímicas (Fundación Campomar, F.C.E.N.-U.B.A, CONICET, Buenos Aires, Argentina.
authors.forename: R J
authors.initials: RJ
authors.lastname: Rodriguez-Suarez
authors.forename: S
authors.initials: S
authors.lastname: Mora-García
authors.forename: R A
authors.initials: RA
authors.lastname: Wolosiuk
mesh_terms: D000595:Amino Acid Sequence; D001937:Brassica / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D002736:Chloroplasts / Q000201:enzymology*; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004789:Enzyme Activation; D004795:Enzyme Stability; D006597:Fructose-Bisphosphatase / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction
chemical_list: D006597:Fructose-Bisphosphatase; D003545:Cysteine