PARENT SESSION

Symposium S3C C3 and Rubisco
Tuesday August 31st, 2004 10:20 AM-12:20 PM Room 510A
Chair: Michael Salvucci
Co-Chair: Akiho Yokota

Structure-function relationship between rubisco-like protein of Bacillus subtilis and photosynthetic rubisco. Yohtaro Saito^*,1, Agnieszka Sekowska², Hiroki Ashida¹, Antoine Danchin², Akiho Yokota¹, ¹ Nara Institute of Science and Technology (NAIST), Ikoma-city, Nara, Japan² Genetics of Bacterial Genomes Institut Pasteur, France

ABSTRACT- Phylogenetic analysis of rbcL DNA sequences divides ribulose-1,5-bisphasphate carboxylase/oxygenase (RuBisCO) into four major clades. Forms I through III include the amino acid residues that are required for catalytic activity of RuBisCO and are capable of catalyzing both the carboxylation and oxygenation of ribulose-1,5-bisphasphate (RuBP). Form IV lacks the residues involved in binding of the phosphate group on C5 and the functional loop 6 in photosynthetic RuBisCO, and has no carboxylation and oxygenation activity. Form IV is thereby referred to as the RuBisCO-like protein (RLP). We found that RLP from Bacillus subtilis catalyzed the 2,3-diketo-5-methylthiopentyl-1-phosphate enolase reaction in the methionine salvage pathway. This reaction resembles that of the first step in the RuBisCO reaction. The first step on the reaction pathway is the tautomerisation of RuBP to its enediol(ate); the hydrogen at C3 is withdrawn by carbamylated Lys-201, and a double bond is formed between C2 and C3. We created several mutated RLPs where a few lysine residues essential for RuBisCO and some residues were changed to other amino acid residues. The RLP-disrupted mutant of B. subtilis can not use methylthioribose (MTR) as the sulfur source. Some mutated RLPs were able to complement RLP deletion mutant. K150E and K175E (corresponding to K175 and K201, respectively, in spinach RuBisCO) could not support MTR-dependent growth. Recombinant mutated RLPs were expressed in Escherichia coli and purified. The catalytic activities of K150E and K175E were less than 1% of that of the wild type. Some other residues were involved in the catalysis of RLP. Thus, well conserved, catalytic lysines of RuBisCO also play key roles in RLP.

KEY WORDS: rubisco-like protein, site-directed mutagenesis, rubisco, active site