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J. Biol. Chem., Vol. 282, Issue 6, 3809-3818, February 9, 2007

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Linked Rubisco Subunits Can Assemble into Functional Oligomers without Impeding Catalytic Performance^*

From the Molecular Plant Physiology, Research School of Biological Sciences, Australian National University, P. O. Box 475, Canberra, Australian Capital Territory 2601, Australia

Although transgenic manipulation in higher plants of the catalytic large subunit (L) of the photosynthetic CO₂-fixing enzyme ribulose 1,5-bisphospahte carboxylase/oxygenase (Rubisco) is now possible, the manipulation of its cognate small subunit (S) is frustrated by the nuclear location of its multiple gene copies. To examine whether L and S can be engineered simultaneously by fusing them together, the subunits from Synechococcus PCC6301 Rubisco were tethered together by different linker sequences, producing variant fusion peptides. In Escherichia coli the variant PCC6301 LS fusions assembled into catalytically functional octameric ([LS]₈) and hexadecameric ([[LS]₈]₂) quaternary structures that excluded the integration of co-expressed unfused S. Assembly of the LS fusions into Rubisco complexes was impaired 50–90% relative to the assembly of unlinked L and S into L₈S₈ enzyme. Assembly in E. coli was not emulated using tobacco SL fusions that accumulated entirely as insoluble protein. Catalytic measurements showed the CO₂/O₂ specificity, carboxylation rate, and Michaelis constants for CO₂ and ribulose 1,5-bisphosphate for the cyanobacterial Rubisco complexes comprising fusions where the S was linked to the N terminus of L closely matched those of the wild-type L₈S₈ enzyme. In contrast, the substrate affinities and carboxylation rate of the Rubisco complexes comprising fusions where L was fused to the N terminus of S or a six-histidine tag was appended to the C terminus of L were compromised. Overall this work provides a framework for implementing an alternative strategy for exploring simultaneous engineering of modified, or foreign, Rubisco L and S subunits in higher plant plastids.

Received for publication, November 10, 2006 , and in revised form, December 6, 2006.

^* This work was funded by Australian Research Council Discovery Grant DP0450564. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.

¹ To whom correspondence should be addressed. Tel.: 61-2-6125-5073; Fax: 61-2-6125-5075; E-mail: spencer.whitney{at}anu.edu.au.

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