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-methyltransferase (EC 1.2.1.68) was expressed in an active form in Escherichia coli using pET-23 vector. Two steps were used to purify (Phenyl Sepharose and S-adenosylhomocysteine-agarose chromatographies) enzyme to homogeneity. O-Methyl-transferase has a subunit of 40 kDa and native gradient gel electrophoresis indicated the active form is a dimer. Substrate specificity was investigated using over 20 phenolic compounds, which defined the nature of the substrate binding site and required substrate 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-adenosyl-L-methionine must bind before phenolic substrate; however, S-adenosyl-L-homocysteine and phenolic substrate or product can form stable complexes complicating the kinetic mechanism. The role of thiol side chain(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 are invariant among higher plant O-methyltransferases of this class. These residues were replaced by serine and alanine, singly and in combination, using site-directed mutagenesis. All combinations of cysteine replacements at positions 276 and 283 yielded enzyme virtually as active as wild-type and all were still sensitive to thiol inhibition. We concluded 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 surface which sterically hindered the active site.
Meng
H
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Phytotechnology Research Center, Michigan Technological University, Houghton, Michigan 49931-1295, USA.
Campbell
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WH
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013696:Temperature; D014197:Trees / Q000201:enzymology* / Q000235:genetics*
D017931:DNA Primers; D004274:DNA, Recombinant; D008780:Methyltransferases; C019608:caffeate O-methyltransferase