Abstract
Despite conservation of three-dimensional structure and active-site residues, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) enzymes from divergent species differ with respect to catalytic efficiency and CO2/O2 specificity. A deeper understanding of the structural basis for these differences may provide a rationale for engineering an improved enzyme, thereby leading to an increase in photosynthetic CO2 fixation and agricultural productivity. By comparing 500 active-site large subunit sequences from flowering plants with that of the green alga Chlamydomonas reinhardtii, a small number of residues were found to differ in regions previously shown by mutant screening to influence CO2/O2 specificity. When directed mutagenesis and chloroplast transformation were used to change Chlamydomonas Met-42 and Cys-53 to land plant Val-42 and Ala-53 in the large subunit N-terminal domain, little or no change in Rubisco catalytic properties was observed. However, changing Chlamydomonas methyl-Cys-256, Lys-258, and Ile-265 to land plant Phe-256, Arg-258, and Val-265 at the bottom of the alpha/beta-barrel active site caused a 10% decrease in CO2/O2 specificity, largely due to an 85% decrease in carboxylation catalytic efficiency (Vmax/Km). Because land plant Rubisco enzymes have greater CO2/O2 specificity than the Chlamydomonas enzyme, this group of residues must be complemented by other residues that differ between Chlamydomonas and land plants. The Rubisco x-ray crystal structures indicate that these residues may reside in a variable loop of the nuclear-encoded small subunit, more than 20 A away from the active site.
Highlights
□S The on-line version of this article contains a supplemental table showing residues that differ between Chlamydomonas and ϳ500 flowering plant sequences for the Rubisco large subunit
Because land plant Rubisco enzymes have greater CO2/O2 specificity than the Chlamydomonas enzyme, this group of residues must be complemented by other residues that differ between Chlamydomonas and land plants
The loops between -strands and ␣-helixes of the C-terminal ␣/-barrel domain contribute the majority of catalytically essential residues to the active site, but several active-site residues reside in the N-terminal domain of an adjacent large subunit
Summary
Strains and Culture Conditions—C. reinhardtii 2137 mtϩ is the wild-type strain [26]. Photosynthesis-deficient, acetate-requiring mutants 18-7G mtϩ and 25B1 mtϩ were used as hosts for chloroplast transformation. Mutant 18-7G results from an rbcL UAG nonsense mutation that terminates large subunit translation after residue Thr-65 [27]. Mutant 25B1 was created by inserting 480 bp of yeast DNA at a PstI site in the 3Ј coding region of rbcL [28]. Both mutant strains fail to accumulate Rubisco holoenzyme [27, 28]. All strains were maintained at 25 °C in darkness with 10 mM acetate medium containing 1.5% Bacto-agar [26].
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