Two Residues of Rubisco Activase Involved in Recognition of the Rubisco Substrate

Rubisco activase is an AAA+ protein, a superfamily with members that use a “Sensor 2” domain for substrate recognition. To determine whether the analogous domain of activase is involved in recognition of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39), two chimeric activases w...

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Bibliographic Details
Published in:The Journal of biological chemistry 2005-07, Vol.280 (26), p.24864-24869
Main Authors: Li, Cishan, Salvucci, Michael E., Portis, Archie R.
Format: Article
Language:English
Subjects:
Adenosine Triphosphatases - chemistry
Amino Acid Sequence
Dimerization
DNA, Complementary - metabolism
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed
Mutation
Nicotiana - enzymology
Plant Proteins - chemistry
Plant Proteins - genetics
Point Mutation
Protein Binding
Protein Structure, Tertiary
Recombinant Fusion Proteins - chemistry
Ribulose-Bisphosphate Carboxylase - metabolism
Sequence Homology, Amino Acid
Spinacia oleracea - enzymology
Substrate Specificity
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Summary:Rubisco activase is an AAA+ protein, a superfamily with members that use a “Sensor 2” domain for substrate recognition. To determine whether the analogous domain of activase is involved in recognition of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39), two chimeric activases were constructed, interchanging a Sensor 2-containing region between activases from spinach and tobacco. Spinach chimeric activase was a poor activator of both spinach and tobacco Rubisco. In contrast, tobacco chimeric activase activated spinach Rubisco far better than tobacco Rubisco, similar to spinach activase. A point mutation, K311D, in the Sensor 2 domain of the tobacco chimeric activase abolished its ability to better activate spinach Rubisco. The opposite mutation, D311K, in wild type tobacco activase produced an enzyme that activated both spinach and tobacco Rubisco, whereas a second mutation, D311K/L314V, shifted the activation preference toward spinach Rubisco. The involvement of these two residues in substrate selectivity was confirmed by introducing the analogous single and double mutations in cotton activase. The ability of the two tobacco activase mutants to activate wild type and mutant Chlamydomonas Rubiscos was also examined. Tobacco D311K activase readily activated wild type and P89R but not D94K Rubisco, whereas the tobacco L314V activase only activated D94K Rubisco. The tobacco activase double mutant D311K/L314V activated wild type Chlamydomonas Rubisco better than either the P89R or D94K Rubisco mutants, mimicking activation by spinach activase. The results identified a substrate recognition region in activase in which two residues may directly interact with two residues in Rubisco.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M503547200