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

Abstract

ABSTRACT 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 Δ3, 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 α-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 Δ3. 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 α-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.