Abstract
A temperature-conditional, photosynthesis-deficient mutant of the green alga Chlamydomonas reinhardtii, previously recovered by genetic screening, results from a leucine 290 to phenylalanine (L290F) substitution in the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC ). Rubisco purified from mutant cells grown at 25 degrees C has a reduction in CO(2)/O(2) specificity and is inactivated at lower temperatures than those that inactivate the wild-type enzyme. Second-site alanine 222 to threonine (A222T) or valine 262 to leucine (V262L) substitutions were previously isolated via genetic selection for photosynthetic ability at the 35 degrees C restrictive temperature. These intragenic suppressors improve the CO(2)/O(2) specificity and thermal stability of L290F Rubisco in vivo and in vitro. In the present study, directed mutagenesis and chloroplast transformation were used to create the A222T and V262L substitutions in an otherwise wild-type enzyme. Although neither substitution improves the CO(2)/O(2) specificity above the wild-type value, both improve the thermal stability of wild-type Rubisco in vitro. Based on the x-ray crystal structure of spinach Rubisco, large subunit residues 222, 262, and 290 are far from the active site. They surround a loop of residues in the nuclear-encoded small subunit. Interactions at this subunit interface may substantially contribute to the thermal stability of the Rubisco holoenzyme.
Highlights
Holoenzyme at apparently normal rates [4], the reduced level of Rubisco must result from increased degradation of an unstable holoenzyme in vivo (4 – 6)
Mutant Recovery and Phenotypes—When the rbcL-25B1 insertion mutant was transformed with either pLS-alanine 222 to threonine (A222T) or pLS-valine 262 to leucine (V262L), photosynthesis-competent colonies were recovered on minimal medium in the light at a frequency of 1.2 ϫ 10Ϫ6 cells
Because this transformation frequency was comparable to the frequency obtained with wildtype rbcL [31], it was evident that the A222T and V262L suppressor mutations alone had no substantial deleterious effect on Rubisco function
Summary
2.2 a The values are the means Ϯ S.D. of three separate enzyme preparations. b Calculated values. 2.2 a The values are the means Ϯ S.D. of three separate enzyme preparations. 3.7 3.2 3.0 3.3 4.2 4.1 subunit substitutions increased the carboxylation kcat, CO2/O2 specificity, and thermal stability of L290F mutant Rubisco [8], and are relatively far from the active site [7], we were curious to see the effect of these substitutions on an otherwise wildtype enzyme
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