CHARACTERIZATION AND SITE-DIRECTED MUTAGENESIS OF ASPEN LIGNIN-SPECIFIC O-METHYLTRANSFERASE EXPRESSED IN ESCHERICHIA-COLI

Aspen lignin-specific caffeic acid/5-hydroxyferulic acid 3/5-O-methylt ransferase (EC 1.2.1.68) was expressed in an active form in Escherichi a coli using pET-23 vector. Two steps were used to purify (Phenyl Seph arose and S-adenosylhomocysteine-agarose chromatographies) enzyme to h omogeneity. O-Methyl-transferase has a subunit of 40 kDa and native gr adient gel electrophoresis indicated the active form is a dimer. Subst rate specificity was investigated using over 20 phenolic compounds, wh ich defined the nature of the substrate binding site and required subs trate characteristics such as a hydroxyl group para to the side chain. Enzyme accommodates large substrates well if the side chain contains the trans-double bond found in lignin precursors. Kinetically S-adenos yl-L-methionine must bind before phenolic substrate; however, S-adenos yl-L-homocysteine and phenolic substrate or product can form stable co mplexes complicating the kinetic mechanism. The role of thiol side cha in(s) in the catalytic mechanism was investigated since the enzyme is inhibited by p-chloromercuribenzoate. Of nine cysteine residues in the enzyme's sequence, only cysteine residues at positions 276 and 283 ar e invariant among higher plant O-methyltransferases of this class. The se residues were replaced by serine and alanine, singly and in combina tion, using site-directed mutagenesis, All combinations of cysteine re placements at positions 276 and 283 yielded enzyme virtually as active as wild-type and all were still sensitive to thiol inhibition. We con cluded that thiol(s) were not important in the catalytic mechanism of this class of O-methyltransferases and sensitivity to the large thiol inhibitor was probably due to reaction of cysteine thiol(s) near the s urface which sterically hindered the active site. (C) 1996 Academic Pr ess, Inc.