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J. Biol. Chem., Vol. 280, Issue 26, 24864-24869, July 1, 2005

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

Cishan Li, Michael E. Salvucci, and Archie R. Portis, Jr.¶||

From the Department of Plant Biology, University of Illinois, Urbana, Illinois 61801, Western Cotton Research Laboratory, Agricultural Research Service, United States Department of Agriculture, Phoenix, Arizona 85040 and ^¶Photosynthesis Research Unit, Agricultural Research Service, United States Department of Agriculture, Urbana, Illinios 61801

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.

Received for publication, March 31, 2005 , and in revised form, April 28, 2005.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed: Photosynthesis Research Unit, Agricultural Research Service, United States Dept. of Agriculture, Urbana IL 61801. Tel.: 217-244-3083; Fax: 217-244-4419; E-mail: arportis{at}uiuc.edu.

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