Phosphoribulokinase (PRK), unique to photosynthetic organisms, is regulated in higher plants by thioredoxin-mediated thiol-disulfide exchange in a light-dependent manner. Prior attempts to overexpress the higher plant PRK gene in Escherichia coli for structure-function studies have been hampered by sensitivity of the recombinant protein to proteolysis as well as toxic effects of the protein on the host. To overcome these impediments, we have spliced the spinach PRK coding sequence immediately downstream from the AOX1 (alcohol oxidase) promoter of Pichia pastoris, displacing the chromosomal AOX1 gene. The PRK gene is now expressed, in response to methanol, at 4-6% of total soluble protein, without significant in vivo degradation of the recombinant enzyme. This recombinant spinach PRK is purified to homogeneity by successive anion-exchange and dye-affinity chromatography and is shown to be electrophoretically and kinetically indistinguishable from the authentic spinach counterpart. Site-specific replacement of all of PRK's cysteinyl residues (both individually and in combination) demonstrates a modest catalytically facilitative role for Cys-55 (one of the regulatory residues) and the lack of any catalytic role for Cys-16 (the other regulatory residue), Cys-244, or Cys-250. Mutants with seryl substitutions at position 55 display non-hyperbolic kinetics relative to the concentration of ribulose 5-phosphate. Sulfate restores hyperbolic kinetics and enhances kinase activity, presumably reflecting conformational differences between the position 55 mutants and wild-type enzyme. Catalytic competence of the C16S-C55S double mutant proves that mere loss of free sulfhydryl groups by oxidative regulation cannot account entirely for the accompanying total inactivation.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				A. K. Michels, N. Wedel, and P. G. Kroth Diatom Plastids Possess a Phosphoribulokinase with an Altered Regulation and No Oxidative Pentose Phosphate Pathway Plant Physiology, March 1, 2005; 137(3): 911 - 920. [Abstract] [Full Text] [PDF]

				S. Lebreton, E. Graciet, and B. Gontero Modulation, via Protein-Protein Interactions, of Glyceraldehyde-3-phosphate Dehydrogenase Activity through Redox Phosphoribulokinase Regulation J. Biol. Chem., March 28, 2003; 278(14): 12078 - 12084. [Abstract] [Full Text] [PDF]

				D. Kobayashi, M. Tamoi, T. Iwaki, S. Shigeoka, and A. Wadano Molecular Characterization and Redox Regulation of Phosphoribulokinase from the Cyanobacterium Synechococcus sp. PCC 7942 Plant Cell Physiol., March 15, 2003; 44(3): 269 - 276. [Abstract] [Full Text] [PDF]

				R. Entus, M. Poling, and K. M. Herrmann Redox Regulation of Arabidopsis 3-Deoxy-D-arabino-Heptulosonate 7-Phosphate Synthase Plant Physiology, August 1, 2002; 129(4): 1866 - 1871. [Abstract] [Full Text] [PDF]

				M. K. Geck and F. C. Hartman Kinetic and Mutational Analyses of the Regulation of Phosphoribulokinase by Thioredoxins J. Biol. Chem., June 9, 2000; 275(24): 18034 - 18039. [Abstract] [Full Text] [PDF]