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. 1991 Nov;97(3):913–920. doi: 10.1104/pp.97.3.913

Partial Purification and Characterization of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Large Subunit εN-Methyltransferase 1

Robert L Houtz 1,2, Malcolm Royer 1,2, Michael E Salvucci 1,2
PMCID: PMC1081104  PMID: 16668531

Abstract

The large subunit (LS) of tobacco (Nicotiana rustica) ribulose-1,5-bisphosphate carboxylase/oxygenase (ribulose-P2 carboxylase) contains a trimethyllysyl residue at position 14, whereas this position is unmodified in spinach ribulose-P2 carboxylase. A protein fraction was isolated from tobacco chloroplasts by rate-zonal centrifugation and anion-exchange fast protein liquid chromatography that catalyzed transfer of methyl groups from S-adenosyl-[methyl-3H]-l-methionine to spinach ribulose-P2 carboxylase. 3H-Methyl groups incorporated into spinach ribulose-P2 carboxylase were alkaline stable but could be removed by limited tryptic proteolysis. Reverse-phase high-performance liquid chromatography of the tryptic peptides released after proteolysis showed that the penultimate N-terminal peptide from the LS of spinach ribulose-P2 carboxylase contained the site of methylation, which was identified as lysine-14. Thus, the methyltransferase activity can be attributed to S-adenosylmethionine:ribulose-P2 carboxylase LS (lysine) `N-methyltransferase, a previously undescribed chloroplast enzyme. The partially purified enzyme was specific for ribulose-P2 carboxylase and exhibited apparent Km values of 10 micromolar for S-adenosyl-l-methionine and 18 micromolar for ribulose-P2 carboxylase, a Vmax of 700 picomoles CH3 groups transferred per minute per milligram protein, and a broad pH optimum from 8.5 to 10.0. S-Adenosylmethionine:ribulose-P2 carboxylase LS (lysine)εN-methyltransferase was capable of incorporating 24 3H-methyl groups per spinach ribulose-P2 carboxylase holoenzyme, forming 1 mole of trimethyllysine per mole of ribulose-P2 carboxylase LS, but was inactive on ribulose-P2 carboxylases that contain a trimethyllysyl residue at position 14 in the LS. The enzyme did not distinguish between activated (Mg2+ and CO2) and unactivated forms of ribulose-P2 carboxylase as substrates. However, complexes of activated ribulose-P2 carboxylase with the reaction-intermediate analogue 2′-carboxy-d-arabinitol-1,5-bisphosphate, or unactivated spinach ribulose-P2 carboxylase with ribulose-1,5-bisphosphate, were poor substrates for tobacco LS εN-methyltransferase.

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Selected References

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