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Related Experiment Videos

Directed mutagenesis of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase. Loop 6 substitutions complement

G Zhu1, R J Spreitzer

  • 1Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588-0664, USA.

The Journal of Biological Chemistry
|August 2, 1996
PubMed
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Investigating chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) structure, this study found that specific amino acid changes in loop 6 significantly impact its CO2/O2 specificity and holoenzyme stability.

Area of Science:

  • Biochemistry
  • Plant Molecular Biology
  • Enzyme Kinetics

Background:

  • The active-site loop 6 structure is critical for the CO2/O2 specificity of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco).
  • Chlamydomonas reinhardtii Rubisco exhibits lower CO2/O2 specificity compared to higher plant Rubisco, potentially due to sequence variations in loop 6.

Purpose of the Study:

  • To investigate whether sequence differences in loop 6 account for variations in Rubisco's catalytic efficiency and CO2/O2 specificity.
  • To analyze the roles of conserved residues Leu-326 and Met-349 at the base of loop 6 in C. reinhardtii Rubisco.

Main Methods:

  • In vitro mutagenesis was used to create L326I and M349L substitutions in the Rubisco large subunit of C. reinhardtii.
  • Chloroplast transformation was employed to assess the in vivo effects of these mutations.

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  • Enzyme activity, holoenzyme stability, and CO2/O2 specificity were measured for the mutated Rubisco variants.

    • Main Results:

    • The M349L substitution had minimal impact on holoenzyme stability and specificity.
    • The L326I substitution destabilized the Rubisco holoenzyme both in vitro and in vivo.
    • The combined L326I and M349L substitutions reduced CO2/O2 specificity by 21% and Vmax of carboxylation by 74%, while partially alleviating instability.

    Conclusions:

    • Interactions involving loop 6 residues are crucial for maintaining holoenzyme stability and catalytic efficiency in higher plant Rubisco.
    • Specific amino acid substitutions at position 326 significantly affect Rubisco stability and function.
    • Structural differences in loop 6 contribute to the distinct catalytic properties of C. reinhardtii Rubisco compared to higher plants.