Reduced CO₂/O₂ specificity of ribulose-bisphosphate carboxylase/oxygenase in a temperature-sensitive chloroplast mutant of Chlamydomonas

Authors

Zhixiang Chen, University of Nebraska at Lincoln
Chris J. Chastain, Texas A & M University - College Station
Souhail R. Al-Abed, University of Nebraska - Lincoln
Raymond Chollet, University of Nebraska - LincolnFollow
Robert J. Spreitzer, University of Nebraska - LincolnFollow

Date of this Version

1988

Comments

Published in Proc. Nati. Acad. Sci. USA Vol. 85, pp. 4696-4699, July 1988. Used by Permission

Abstract

The Chlamydomonas reinhardtii chloroplast mutant 68-4PP is phenotypically indistinguishable from wild type at 25°C but fails to grow photosynthetically at 35°C. It had about 30% of the wild-type level of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) holoenzyme and carboxylase activity when grown at 25°C, but less than 15% when grown at 35°C. Pulse-labeling with ³⁵S showed that the decrease in enzyme level at the restrictive temperature was not a result of reduced synthesis of enzyme subunits. The CO₂/O₂ specificity factor (V_cK_o/V_oK_c, where V_c and V_o are V_max values for carboxylation and oxygenation and K_c and K_o are K_m values for CO₂ and 0₂) of the mutant enzyme was found to be significantly less than that of the wild-type enzyme (54 ± 2 and 62 ± 1, respectively), and this alteration was accompanied by increases in K_o and K_c and a decrease in V_c/V_o. DNA sequencing revealed a single missense mutation in the 684PP chloroplast large-subunit gene. This mutation causes leucine to be replaced by phenylalanine at position 290 in the large subunit polypeptide sequence. These results (i) support previous studies that implicated this region of the large subunit as an important structural component of the enzyme's function and (ii) demonstrate that chloroplast genetic modification of the CO₂/O₂ specificity factor of a plant-type carboxylase/oxygenase is feasible.