EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF RECOMBINANT RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE OXYGENASE LARGE SUBUNIT N-EPSILON-METHYLTRANSFERASE/

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subuni t (LS) N-6-methyltransferase (Rubisco LSMT, EC 2.1.1.127) catalyzes me thylation of the LS of Rubisco. A pea (Pisum sativum L. cv Laxton's Pr ogress 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 leve l of soluble Rubisco LSMT and the associated enzymatic activity. Howev er, the soluble form of Rubisco LSMT occurred as two molecular mass fo rms 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 38 amino acids of Rubisco LSMT resulted in a 16-fold increase in solubility and activity. Further addition of a 3' nucleotide seque nce coding for a hexahistidyl carboxy-terminal peptide enabled purific ation of the N-terminally truncated Rubisco LSMT to homogeneity. Five milligrams of pure recombinant Rubisco LSMT was obtained from a l-lite r E. coli cell culture. The apparent kinetic constants for recombinant Rubisco LSMT for spinach Rubisco and AdoMet were only slightly differ ent from the constants determined using affinity-purified native Rubis co LSMT from pea chloroplasts. However, there was a 6- to 7-fold reduc tion in the kappa(cat) for Rubisco LSMT, which was apparently a conseq uence of catalytic inactivation due to exposure to NiSO4 during purifi cation. The availability of larger quantities of purified Rubisco LSMT should enable studies of the structure-function relationships in Rubi sco LSMT and moreover its interaction with Rubisco. (C) 1998 Academic Press.