Neish Constitution and Biosynthesis of Lignin.

Freudenberg’s method of pursuing different analytical and synthetical pathways simultaneously revealed its value when he brought together this accumulated expertise in order to determine the structure of lignin, an extremely complicated natural polymer responsible for the stability of vascular plants and therefore one of the most abundant natural products. However, its separation and chemical treatment presented major problems. New analytical methods, degradation procedures, and model syntheses had to be developed. They supported Freudenberg’s assumption that coniferyl alcohol was a major precursor of lignin, an assumption he was able to confirm by simulating the enzymatic lignification in the laboratory, interrupting the polymerization, and identifying the oligomeric intermediates. For almost three decades, however, progress on the lignin problem was disappointingly slow. In 1952 Freudenberg achieved the first real breakthrough when he succeeded in isolating the oligomers, determining their structure, and explaining the mechanism of their formation through intermediate radicals. Lignin then became the main field of his research, which from 1962 to 1965 culminated in the design of a formula scheme for the constitution of spruce lignin, involving eighteen C6—C3 units.

Constitution and biosynthesis of lignin. - CAB Direct

Saturation mutagenesis was performed at sites 130, 131, 133, 134,139,164,165, 175,186, 319, 326, and 327 of IEMT following the QuikChange site-directed mutagenesis strategy (Stratagene) using NNK degenerate primers (N represents a mixture of A, T, G, C, and K for G/T) (). The codon NNK has 32-fold degeneracy and encodes all 20 amino acids without rare codons.

Biosynthesis and Constitution of Lignin - Nature

Constitution and Biosynthesis of Lignin

Following a path of directed evolution with one amino acid substitution at a time, and employing iterative site-saturation mutagenesis, we created an efficient, novel monolignol 4-O-methyltransferase from phenylpropene O-methyltransferase. Phenylpropene O-methyltransferases and a few other phenolic O-methyltransferases were demonstrated to have evolved naturally in plants from the lignin biosynthetic enzyme, COMT (,). The evolution of these phenolic O-methyltransferases primarily was archived through gene duplication and subsequent substitutions of only a limited set of amino acid residues. A previous study () and our current homology modeling indicate that IEMT differs from COMT in its putative active site, primarily seven amino acid residues (). Therefore, these distinct active site residues in the two enzymes most likely represent evolutionarily plastic sites that dominate substrate discrimination and regioselective methylation. Further modulating these plastic sites might interrogate and engender the desired novel functionalities. By saturation mutagenesis, during which we introduced a full set of 20 amino acid substitutions at each of the seven active plastic sites, we demonstrated that two amino acid sites in IEMT, Glu-165 and Thr-133, are critical for substrate discrimination/binding. Substituting both sites with hydrophobic residues enables the resulting variants to effectively recognize and accommodate a monolignol substrate, while retaining the ability for 4-O-methylation (see and ). Other sites tested displayed a lesser effect, or none, in initiating the novel substrate preference of the enzyme. Subsequent iterative mutations using the single mutant variants from both sites created the double mutations with higher activity. Serendipitously, these double mutants combine the beneficial substitutions of Glu-165 and Thr-133. These apparent additive mutation effects support the notion that the targeted property in directed enzyme evolution can be acquired through a series of single beneficial mutations, and that combination of the single mutations would retain the desired properties (). After three rounds of saturation mutations, variant T133L/E165I/F175I showed adequate catalytic capacity in the 4-O-methylation of monolignols; its catalytic efficiency and binding affinity to monolignols, respectively, were more than 70- and 13-fold higher than those of the wild-type enzyme. Interestingly, these three site mutations created an apparently novel substrate binding pocket for accommodating monolignols (D), pointing to the facile structural plasticity of phenolic OMTs in the evolution of new biochemical functions.