Expression, purification, and characterization of recombinant ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit nepsilon-methyltransferase.
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) Nepsilon-methyltransferase (Rubisco LSMT, EC 2.1.1.127) catalyzes methylation of the LS of Rubisco. A pea (Pisum sativum L. cv Laxton's Progress No. 9) Rubisco LSMT cDNA was expressed in Escherichia coli, but most of the expressed protein was found in the insoluble fraction as an inclusion body. Expression at lower temperatures increased the level of soluble Rubisco LSMT and the associated enzymatic activity. However, the soluble form of Rubisco LSMT occurred as two molecular mass forms with the lower molecular mass suggestive of N-terminal processing at Ser-37. Deletion of 108 nucleotides from the 5' end encoding the N-terminal 36 amino acids of Rubisco LSMT resulted in a 10-fold increase in solubility and activity. Further addition of a 3' nucleotide sequence coding for a hexahistidyl carboxy-terminal peptide enabled purification of the N-terminally truncated Rubisco LSMT to homogeneity. Five milligrams of pure recombinant Rubisco LSMT was obtained from a 1-liter E. coli cell culture. The apparent kinetic constants for recombinant Rubisco LSMT for spinach Rubisco and AdoMet were only slightly different from the constants determined using affinity-purified native Rubisco LSMT from pea chloroplasts. However, there was a 6- to 7-fold reduction in the kcat for Rubisco LSMT, which was apparently a consequence of catalytic inactivation due to exposure to NiSO4 during purification. The availability of larger quantities of purified Rubisco LSMT should enable studies of the structure-function relationships in Rubisco LSMT and moreover its interaction with Rubisco.
Plant Physiology/Biochemistry/Molecular Biology Program, University of Kentucky, Lexington, Kentucky, 40546, USA.
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Q
Zheng
E J
EJ
Simel
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PE
Klein
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MT
Royer
R L
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Houtz
D000595:Amino Acid Sequence; D001483:Base Sequence; D002736:Chloroplasts / Q000201:enzymology; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D011495:Histone-Lysine N-Methyltransferase / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D018532:Pisum sativum / Q000201:enzymology / Q000235:genetics; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013379:Substrate Specificity
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; C097344:ribulose-1,5-bisphosphate carboxylase-oxygenase large subunit epsilonN-methyltransferase; D011495:Histone-Lysine N-Methyltransferase