Directed mutagenesis of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase. Loop 6 substitutions complement for structural stability but decrease catalytic efficiency.
The structure of active-site loop 6 plays a role in determining the CO2/O2 specificity of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39). Rubisco from the green alga Chlamydomonas reinhardtii differs from higher plant Rubisco within the loop 6 region, and the C. reinhardtii enzyme has a CO2/O2 specificity 25% lower than that of higher plant enzymes. To examine whether differences in sequence may account for differences in catalytic efficiency, we focused on a conserved pair of residues that are in van der Waals contact at the base of loop 6. C. reinhardtii Rubisco contains Leu-326 and Met-349, whereas higher plant enzymes contain Ile-326 and Leu-349. By employing in vitro mutagenesis and chloroplast transformation, L326I and M349L substitutions were created within the Rubisco large subunit of C. reinhardtii. M349L had little effect, but L326I destabilized the holoenzyme in vivo and in vitro. When present together, the M349L substitution partially alleviated the instability resulting from the L326I substitution, but caused a 21% decrease in CO2/O2 specificity and a 74% decrease in the Vmax of carboxylation. Interactions between loop 6 and other structural regions are likely to be responsible for both holoenzyme stability and catalytic efficiency in higher plant Rubisco enzymes.
Zhu
G
G
Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588-0664, USA.
Spreitzer
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D016825:Chlamydomonas reinhardtii / Q000201:enzymology / Q000235:genetics; D002736:Chloroplasts / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D017433:Protein Structure, Secondary; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D012273:Ribulose-Bisphosphate Carboxylase