Chloroplast fructose 1,6-bisphosphatase with changed redox modulation: comparison of the Galdieria enzyme with cysteine mutants from spinach

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• 作者：Reichert ; Angelika ; Dennes ; André ; Vetter ; Susanne ; Scheibe ; Renate
• 关键词：Chloroplast enzyme ; Fructose 1 ; 6-bisphosphatase ; Redox modulation ; Site-directed mutagenesis ; Spinacia oleracea ; Galdieria sulphuraria ; FBPase ; fructose 1 ; 6-bisphosphatase ; IPTG ; isopentenyl thio-galactoside ; IEP ; isoelectric point
• 刊名：Biochimica et Biophysica Acta - Proteins and Proteomics
• 出版年：2003
• 出版时间：February 21, 2003
• 年：2003
• 卷：1645
• 期：2
• 页码：212-217
• 全文大小：234 K

文摘

Spinach fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11), a redox-modulated chloroplast enzyme and part of the Calvin cycle, and three different Cys mutants were expressed in E. coli. The properties of the purified proteins were compared to those of native and recombinant chloroplast FBPase from the red alga Galdieria sulphuraria. In spinach chloroplast FBPase, Cys¹⁵⁵ and Cys¹⁷⁴ are engaged in the formation of the disulfide bridge. The corresponding mutants are active when expressed in E. coli, while C179S is inactive and can be reductively activated as can the wild-type enzyme. The active C174S mutant, however, could be inactivated by oxidation, and reactivated, but only by reduction, not alternatively with high pH and high Mg²⁺ as is the case for the wild-type enzyme. In the sequence of Galdieria FBPase, the Cys that corresponds to Cys¹⁷⁹ in the spinach enzyme is lacking. However, the Galdieria FBPase, in contrast to the spinach Cys¹⁷⁹ mutant, does not show any indication for a comparable redox modulation of its activity. Instead, oxidation only leads to partial inactivation without any qualitative changes in enzyme properties. Upon reduction, the lost activity can be recovered.