TWO RESIDUES OF RUBISCO ACTIVASE INVOLVED IN RECOGNITION OF THE RUBISCO SUBSTRATE.

Authors: LI, CISHAN - UNIVERSITY OF ILLINOIS; Salvucci, Michael; Portis Jr, Archie

Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/2/2005
Publication Date: 7/1/2005
Citation: Li, C., Salvucci, M.E., Portis Jr, A.R. 2005. Two residues of Rubisco activase involved in recognition of the Rubisco substrate. Journal of Biological Chemistry. 280(26):24864-24869.

Interpretive Summary: The activity of Rubisco, the enzyme that captures carbon dioxide, often limits photosynthesis, the process by which plants use light energy from the sun to make carbohydrates for growth from carbon dioxide and water. Rubisco activity is maintained by another chloroplast protein, Rubisco activase. Rubisco activity might be increased to improve plant growth by altering its interaction with Rubisco. Past work showed that neither Rubisco nor activase from one species of plants, the Solanaceae, worked well in combination with the other protein from other species. This peculiar specificity preference was shown to be determined by two amino acids on the surface of Rubisco. In this work, we identified a substrate recognition region in activase in which two amino acids may directly interact with the two previously identified amino acids in Rubsisco. This information will benefit scientists attempting to modify the properties of Rubisco and the activase in ways beneficial for increased photosynthesis by crop plants.

Technical Abstract: Rubisco activase is a AAA+ protein, a superfamily with members that use a "Sensor 2" domain for substrate recognition. To determine if 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, while 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, while the tobaco 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 identify a substrate recognition region in activase in which two residues may directly interact with two residues in Rubisco.