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Functional Plant Biology Functional Plant Biology Society
Plant function and evolutionary biology
RESEARCH ARTICLE

Synthesis of Spinach Phosphoribulokinase and Ribulose 1,5-Bisphosphate in Escherichia coli

GS Hudson, MK Morell, YBC Arvidsson and TJ Andrews

Australian Journal of Plant Physiology 19(3) 213 - 221
Published: 1992

Abstract

Phosphoribulokinase catalyses the reaction which supplies the CO2-acceptor of the photosynthetic carbon reduction cycle, D-ribulose 1,5-bisphosphate (RuBP). In plants, this enzyme is inactivated in the dark by oxidation of two cysteinyl residues, Cys16 and Cys55, on the same subunit. A cDNA for spinach phosphoribulokinase was isolated by amplification from leaf RNA and cloned into a bacterial expression plasmid to give plasmid pMMPRK. Escherichia coli carrying pMMPRK grown in rich medium accumulated both the enzyme phosphoribulokinase and its reaction product RuBP. This accumulation of RuBP severely retarded growth of the cells on minimal media. The enzyme was isolated from cell extracts and shown to be kinetically similar to authentic phosphoribulokinase isolated from spinach leaves. Cys55 was altered by site-directed mutagenesis to explore the role of this residue in catalysis and regulation. Alteration of Cys55 to Ser or Gly caused a 10-fold reduction in Vmax and altered Km(ATP). These results are consistent with Cys55 being located at the ATP-binding site but the side chain of this residue is clearly not essential for catalysis. Loss of the Cys55 thiol group accounts for most, but not all, of the loss of activity which occurs during dark inactivation of phosphoribulokinase.

https://doi.org/10.1071/PP9920213

© CSIRO 1992

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