Genkov, Todor , Spreitzer, Robert J. .
Highly-conserved small-subunit residues affect Rubisco function.
It has been difficult to study the role of the small subunit in Rubisco function and assembly because there is a family of rbcS genes in the nucleus that cannot be eliminated. However, in the green alga Chlamydomonas reinhardtii, the family of rbcS genes has been deleted, and this mutant strain can be maintained with acetate as an alternative carbon source. Although the chloroplast-encoded large subunit contains the active site, recent transformation studies have shown that the small subunit can also influence catalysis. In the present study, the ten most-conserved residues of all small subunits were investigated. Previous studies with cyanobacterial Rubisco expressed in E. coli had indicated that several of these residues were essential for assembly. When each of the ten residues was substituted with Ala, it was surprising to find that none blocked the photosynthetic growth of Chlamydomonas. Six of the mutant enzymes have decreases in carboxylation catalytic efficiency (Y32A, E43A, W73A, L78A, P79A, F81A) and two of these have decreases in CO2/O2 specificity (Y32A, E43A). Seven of the conserved residues interact with or are close to alpha-helix 8 or its preceding loop in the alpha/beta barrel that forms the large-subunit active site. Tyr-32 shields polar residues at the carboxyl-terminal end of large-subunit alpha-helix 8 from solvent. A Y32F enzyme (but not Y32D, Y32E, or Y32R) has normal specificity. Glu-43 forms an ionic bond with Arg-187 in alpha-helix 1. An E43D enzyme has improved CO2/O2 specificity relative to E43A. Further analysis of these specific interactions between large and small subunits may help to explain the variation in CO2/O2 specificity observed among Rubisco enzymes from different species. This research is supported by the US DOE.
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1 - University of Nebraska, Department of Biochemistry
2 - University of Nebraska, Department of Biochemistry, Lincoln, Nebraska, 68588-0664
Presentation Type: Plant Biology Abstract
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM