Partial purification and characterization of ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit εN-methyltransferase

Robert L. Houtz, Malcolm Royer, Michael E. Salvucci

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23 Scopus citations


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) εN-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-arabinitol1,5-bisphosphate, or unactivated spinach ribulose-P2 carboxylase with ribulose-1,5-bisphosphate, were poor substrates for tobacco LS εN-methyltransferase.

Original languageEnglish
Pages (from-to)913-920
Number of pages8
JournalPlant Physiology
Issue number3
StatePublished - 1991

ASJC Scopus subject areas

  • Physiology
  • Genetics
  • Plant Science


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