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

The large subunit (LS) of tobacco (Nicotiana rustica) ribulose1,5-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. 3HMethyl 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 V,,,. 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)'N-methyltransferase was capable of incorporating 24 3H-methyl groups per spinach ribulose-P2 carboxylase holoenzyme, forming I 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 C02) 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 'EN-methyltransferase. ' This work was supported by U.S. Department of Agriculture/ Competitive Research Grants Office Grant 89-37262-4482 and Hatch Project KY 00586 to R.L. H. The investigation reported in this paper (No. 90-10-221) is in connection with a project of the Kentucky Agricultural Experiment Station and is published with approval of the Director. Ribulose-P2 carboxylase2 catalyzes the reduction of atmospheric CO2 during photosynthesis. Much is known about the quaternary structure, catalytic mechanism, active site residues, and in vivo regulatory mechanisms for this abundant enzyme (1). The crystal structure of the higher plant enzyme from spinach has been thoroughly analyzed and described in detail (17). The hexadecameric protein is composed of eight nuclear-encoded SS and eight chloroplast-encoded LS which contain the active site. The LS ofribulose-P2 carboxylase from tobacco and muskmelon plants has been shown to be posttranslationally modified by trimethylation of the e-amine group of Lys-14 in the N terminus. However, wheat and spinach ribulose-P2 carboxylase contain an unmodified lysyl residue at position 14 in the LS (14). The significance of this modification and the reason(s) for the species differences are unknown. The N-terminal region of the LS of ribulose-P2 carboxylase is essential for maximum catalytic activity (15), participates in formation of the active site (17, 18), and shows catalytic dependent conformational changes during catalysis as judged by reduced tryptic accessibility of N-terminal lysyl residues (13). Cyt c (lysine) 'N-methyltransferase (7), ribosomal protein (lysine) 'N-methyltransferase (4), histone (lysine) 'N-methyltransferase (33), and calmodulin (lysine) 'N-methyltransferase (10, 30) all show a high degree of protein substrate specificity. Of these 'N-methyltransferases, only calmodulin (lysine) 'Nmethyltransferase (30), and to a lesser extent Cyt c (lysine) 'N-methyltransferase, has been purified and characterized. An S-adenosylmethionine:protein (lysine) 'N-methyltransferase specific for the methylation of Lys-14 in the LS of ribuloseP2 carboxylase should be localized in chloroplasts from plants in which the LS of ribulose-P2 carboxylase contains trimethyllysine-14. By analogy with studies characterizing calmodulin 'N-methyltransferase (30), ribulose-P2 carboxylase with an unmodified lysyl residue at position 14 in the LS should serve as a substrate for the assay, isolation, and characterization of ribulose-P2 carboxylase LS 'N-methyltransferase. In this report a method is described for the assay, partial purifi2Abbreviations: ribulose-P2 carboxylase, ribulose1 ,5-bisphosphate carboxylase-oxygenase; ribulose-P2, ribulose-1 ,5-bisphosphate; LS (SS), the large (small) subunit of ribulose-l,5-bisphosphate carboxylase/oxygenase; AdoMet, S-adenosyl-L-methionine.