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Applied and Environmental Microbiology, October 2007, p. 6254-6261, Vol. 73, No. 19
0099-2240/07/$08.00+0     doi:10.1128/AEM.00044-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Engineering of a Type III Rubisco from a Hyperthermophilic Archaeon in Order To Enhance Catalytic Performance in Mesophilic Host Cells ^,

Shosuke Yoshida, Haruyuki Atomi, and Tadayuki Imanaka^*

Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan

Received 9 January 2007/ Accepted 26 July 2007

The hyperthermophilic archaeon Thermococcus kodakaraensis harbors a type III ribulose 1,5-bisphosphate carboxylase/oxygenase (Rbc_Tk). It has previously been shown that Rbc_Tk is capable of supporting photoautotrophic and photoheterotrophic growth in a mesophilic host cell, Rhodopseudomonas palustris 3, whose three native Rubisco genes had been disrupted. Here, we have examined the enzymatic properties of Rbc_Tk at 25°C and have constructed mutant proteins in order to enhance its performance in mesophilic host cells. Initial sites for mutagenesis were selected by focusing on sequence differences in the loop 6 and -helix 6 regions among Rbc_Tk and the enzymes from spinach (mutant proteins SP1 to SP7), Galdieria partita (GP1 and GP2), and Rhodospirillum rubrum (RR1). Loop 6 of Rbc_Tk is one residue longer than those found in the spinach and G. partita enzymes, and replacing Rbc_Tk loop 6 with these regions led to dramatic decreases in activity. Six mutant enzymes retaining significant levels of Rubisco activity were selected, and their genes were introduced into R. palustris 3. Cells harboring mutant protein SP6 displayed a 31% increase in the specific growth rate under photoheterotrophic conditions compared to cells harboring wild-type Rbc_Tk. SP6 corresponds to a complete substitution of the original -helix 6 of Rbc_Tk with that of the spinach enzyme. Compared to wild-type Rbc_Tk, the purified SP6 mutant protein exhibited a 30% increase in turnover number (k_cat) of the carboxylase activity and a 17% increase in the k_cat/K_m value. Based on these results, seven further mutant proteins were designed and examined. The results confirmed the importance of the length of loop 6 in Rbc_Tk and also led to the identification of specific residue changes that resulted in an increase in the turnover number of Rbc_Tk at ambient temperatures.

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* Corresponding author. Mailing address: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan. Phone: 81-(0)75-383-2777. Fax: 81-(0)75-383-2778. E-mail: imanaka{at}sbchem.kyoto-u.ac.jp

Published ahead of print on 3 August 2007.

Supplemental material for this article may be found at http://aem.asm.org/.

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Applied and Environmental Microbiology, October 2007, p. 6254-6261, Vol. 73, No. 19
0099-2240/07/$08.00+0     doi:10.1128/AEM.00044-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.