Plant-like substitutions in the large-subunit carboxy terminus of Chlamydomonas Rubisco increase CO2/O2 Specificity

Sriram Satagopan,Robert J Spreitzer

doi:10.1186/1471-2229-8-85

Abstract

BackgroundRibulose-1,5-bisphosphate is the rate-limiting enzyme in photosynthesis. The catalytic large subunit of the green-algal enzyme from Chlamydomonas reinhardtii is ~90% identical to the flowering-plant sequences, although they confer diverse kinetic properties. To identify the regions that may account for species variation in kinetic properties, directed mutagenesis and chloroplast transformation were used to create four amino-acid substitutions in the carboxy terminus of the Chlamydomonas large subunit to mimic the sequence of higher-specificity plant enzymes.ResultsThe quadruple-mutant enzyme has a 10% increase in CO2/O2 specificity and a lower carboxylation catalytic efficiency. The mutations do not seem to influence the protein expression, structural stability or the function in vivo.ConclusionOwing to the decreased carboxylation catalytic efficiency, the quadruple-mutant is not a "better" enzyme. Nonetheless, because of its positive influence on specificity, the carboxy terminus, relatively far from the active site, may serve as a target for enzyme improvement via combinatorial approaches.

Highlights

IntroductionThe catalytic large subunit of the green-algal enzyme from Chlamydomonas reinhardtii is ~90% identical to the flowering-plant sequences, they confer diverse kinetic properties
Ribulose-1,5-bisphosphate is the rate-limiting enzyme in photosynthesis
The carboxy terminus of the spinach large subunit is identical in length to that of Chlamydomonas, and the availability of the spinach crystal structure [10] allows structural comparison (Fig. 1)

Summary

Introduction

The catalytic large subunit of the green-algal enzyme from Chlamydomonas reinhardtii is ~90% identical to the flowering-plant sequences, they confer diverse kinetic properties. To identify the regions that may account for species variation in kinetic properties, directed mutagenesis and chloroplast transformation were used to create four amino-acid substitutions in the carboxy terminus of the Chlamydomonas large subunit to mimic the sequence of higher-specificity plant enzymes. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (EC 4.1.1.39) is limited by a low carboxylation rate and competing oxygenase activity that initiates a wasteful photorespiratory pathway leading to the loss of fixed carbon [1]. The green alga Chlamydomonas reinhardtii is an excellent model for studying plant-like Rubisco enzymes comprised of eight large subunits (~55 kDa, coded by the chloroplast rbcL gene) and eight small subunits (~15 kDa, coded by a family of nuclear rbcS genes) [1]. Because Chlamydomonas Rubisco has a faster carboxylation rate and lower Ω value (page number not for citation purposes)

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