Abstract
The temperature-conditional photosynthesis-deficient mutant 68-4PP of Chlamydomonas reinhardtii results from a Leu-290 to Phe substitution in the chloroplast-encoded large subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39). Although this substitution occurs relatively far from the active site, the mutant enzyme has a reduced ratio of carboxylation to oxygenation in addition to reduced thermal stability in vivo and in vitro. In an attempt to understand the role of this region in catalysis, photosynthesis-competent revertants were selected. Two revertants, named R96-4C and R96-8E, were found to arise from second-site mutations that cause V262L and A222T substitutions, respectively. These intragenic suppressor mutations increase the CO2/O2 specificity and carboxylation Vmax back to wild-type values. Based on the crystal structure of the spinach holoenzyme, Leu-290 is not in van der Waals contact with either Val-262 or Ala-222. However, all three residues are located at the bottom of the alpha/beta-barrel active site and may interact with residues of the nuclear encoded small subunits. It appears that amino acid residues at the interface of large and small subunits can influence both stability and catalysis.
Highlights
The Rubisco holoenzyme in the chloroplasts of plants and green algae is composed of eight copies each of large and small subunits
We reasoned that if suppressor mutations could arise in other nuclear genes or elsewhere in the 68-4PP rbcL gene, analysis of such suppressors might help to understand the mode of action of the 68-4PP L290F substitution and, perhaps, the means by which S52-2B acts as a suppressor
DNA sequencing of the R96-8E rbcL gene revealed a G to A transition mutation that would change Ala (GCT) to Thr (ACT) at large subunit residue 222
Summary
Strains and Culture Conditions—C. reinhardtii wild-type 2137 mtϩ [26], mutant 68-4PP mtϩ [21, 22], and revertant strains are maintained at 25 °C in darkness with 10 mM acetate medium containing 1.5% Bacto-agar (Difco) [26]. Dies on minimal medium in the light at 35 °C but survives on minimal medium at 25 °C in the light or on acetate medium at either 25 or 35 °C in the dark [21] This temperature-conditional photosynthesis deficiency results from an L290F substitution in the Rubisco large subunit [22, 23]. Crosses were performed as described previously [21, 26], and temperature-conditional, acetate-requiring progeny were scored by replicaplating dark-grown (25 °C) tetrads to minimal medium in the light at 35 °C. ⍀ was determined by the simultaneous measurement of the carboxylase and oxygenase activities of purified Rubisco (20 g/reaction) with 200 M [1-3H]RuBP (7.2 Ci/mol) and 2 mM NaH14CO3 (5.0 Ci/mol) in 30-min reactions at 25 °C [27, 31]. Other kinetic constants of the purified enzymes were determined as described previously [22]
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