Chimeric Arabidopsis thaliana ribulose-1,5-bisphosphate carboxylase/oxygenase containing a pea small subunit protein is compromised in carbamylation.
A cDNA of pea (Pisum sativum L.) RbcS 3A, encoding a small subunit protein (S) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), has been expressed in Arabidopsis thaliana under control of the cauliflower mosaic virus 35S promoter, and the transcript and mature S protein were detected. Specific antibodies revealed two protein spots for the four Arabidopsis S and one additional spot for pea S. Pea S in chimeric Rubisco amounted to 15 to 18% of all S, as judged by separation on two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels from partially purified enzyme preparations and quantitation of silver-stained protein spots. The chimeric enzyme had 11 +/- 1% fewer carbamylated sites and a 11 +/- 1% lower carboxylase activity than wild-type Arabidopsis Rubisco. Whereas pea S expression, preprotein transport, and processing and assembly resulted in a stable holoenzyme, the chimeric enzyme was reproducibly catalytically less efficient. We suggest that the presence of, on average, one foreign S per holoenzyme is responsible for the altered activity. In addition, higher-plant Rubisco, unlike the cyanobacterial enzyme, seems to have evolved species-specific interactions between S and the large subunit protein that are involved in carbamylation of the active site.
Getzoff
T P
TP
Department of Molecular and Cellular Biology, University of Arizona, Tucson 85721-0088, USA.
Zhu
G
G
Bohnert
H J
HJ
Jensen
R G
RG
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D002219:Carbamates / Q000378:metabolism*; D018076:DNA, Complementary; D015180:Electrophoresis, Gel, Two-Dimensional; D017343:Genes, Plant; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D030821:Plants, Genetically Modified; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D002219:Carbamates; D018076:DNA, Complementary; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D012273:Ribulose-Bisphosphate Carboxylase; C070766:carbamic acid