Displaying all 2 publications

Abstract:
Sort:
  1. Dissection of synechococcus rubisco large subunit sections involved in holoenzyme formation in escherichia coli by combinatorial section swapping and sequence analyses
    Yee Hung Yeap, Teng Wei Koay, Boon Hoe Lim
    Sains Malaysiana, 2018;47:2269-2289.
    Engineering the CO2
    -fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to improve photosynthesis
    has long been sought. Rubisco large subunits (RbcL) are highly-conserved but because of certain undefined sequence
    differences, plant Rubisco research cannot fully utilise the robust heterologous Escherichia coli expression system and its
    GroEL folding machinery. Previously, a series of chimeric cyanobacteria Synechococcus elongatus Rubisco, incorporated
    with sequences from the green alga Chlamydomonas reinhardtii, were expressed in E. coli; differences in RbcL sections
    essential for holoenzyme formation were pinpointed. In this study, the remaining sections, presumably not crucial for
    holoenzyme formation and also the small subunit (RbcS), are substituted to further ascertain the possible destabilising
    effects of multiple section mutations. To that end, combinations of Synechococcus RbcL Sections 1 (residues 1-47), 2
    (residues 48-97), 5 (residues 198-247) and 10 (residues 448-472), and RbcS, were swapped with collinear Chlamydomonas
    sections and expressed in E. coli. Interestingly, only the chimera with Sections 1 and 2 together produces holoenzyme and
    an interaction network of complementing amino acid changes is delineated by crystal structure analysis. Furthermore,
    sequence-based analysis also highlighted possible GroEL binding site differences between the two RbcLs.
    Matched MeSH terms: Ribulosephosphates
  2. Characterization of Chlamydomonas Ribulose-1,5-bisphosphate carboxylase/oxygenase variants mutated at residues that are post-translationally modified
    Rasineni GK, Loh PC, Lim BH
    Biochim Biophys Acta Gen Subj, 2017 Feb;1861(2):79-85.
    PMID: 27816753 DOI: 10.1016/j.bbagen.2016.10.027
    BACKGROUND: Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the chloroplast enzyme that fixes CO2 in photosynthesis, but the enzyme also fixes O2, which leads to the wasteful photorespiratory pathway. If we better understand the structure-function relationship of the enzyme, we might be able to engineer improvements. When the crystal structure of Chlamydomonas Rubisco was solved, four new posttranslational modifications were observed which are not present in other species. The modifications were 4-hydroxylation of the conserved Pro-104 and 151 residues, and S-methylation of the variable Cys-256 and 369 residues, which are Phe-256 and Val-369 in land plants. Because the modifications were only observed in Chlamydomonas Rubisco, they might account for the differences in kinetic properties between the algal and plant enzymes.

    METHODS: Site-directed mutagenesis and chloroplast transformation have been used to test the essentiality of these modifications by replacing each of the residues with alanine (Ala). Biochemical analyses were done to determine the specificity factors and kinetic constants.

    RESULTS: Replacing the modified-residues in Chlamydomonas Rubisco affected the enzyme's catalytic activity. Substituting hydroxy-Pro-104 and methyl-Cys-256 with alanine influenced Rubisco catalysis.

    CONCLUSION: This is the first study on these posttranslationally-modified residues in Rubisco by genetic engineering. As these forms of modifications/regulation are not available in plants, the modified residues could be a means to modulate Rubisco activity.

    GENERAL SIGNIFICANCE: With a better understanding of Rubisco structure-function, we can define targets for improving the enzyme.

    Matched MeSH terms: Ribulosephosphates/genetics*
Related Terms
    Try searching for something
Filters
Contact Us

Please provide feedback to Administrator (afdal@afpm.org.my)

External Links