Constitution and Biosynthesis of Lignin | Karl …

Get this from a library! Constitution and biosynthesis of lignin. [Karl Freudenberg; A C Neish]

Constitution and Biosynthesis of Lignin - Karl …

By modeling the double mutant variant and docking monolignol into its active site (C), we identified a range of additional amino acid sites potentially directly contacting, or proximal to the docked monolignol. They included Leu-139 and Phe-175 that might contribute to constructing a hydrophobic cavity to interact with the 3-methoxy group of the bound compound, as do the corresponding residues in COMT (). Therefore, to optimize enzymatic activity, we imposed further saturation mutations on those recognized sites via T133L/E165F and T133L/E165I as parental templates. Screening the triple mutant libraries, we identified two mutants, T133L/E165I/F175I and T133L/L139Q/E165F, that exhibit a major increase in their specific activity on monolignols (C). Kinetic analysis revealed that the former displays the catalytic efficiency of 1245 and 1463 −1 s−1, respectively, for coniferyl and sinapyl alcohols, namely, more than 70-fold increases compared with the wild-type enzyme ().

Constitution and Biosynthesis of Lignin von Karl Freudenberg, A

Constitution And Biosynthesis Of Lignin - …

The novel enzyme variants we created greatly liberated the highly constrained substrate promiscuity of the parental IEMT. The triple mutants exhibited a broad spectrum of substrate preference on phenolics (D). Many examples of directed evolution have demonstrated that enzymes with promiscuous functions exhibit high plasticity; they might well be used as “generalists” to further tailor particular catalytic properties (,). One of our triple mutants, T133L/L139Q/E165F, catalytically prefers the guaiacyl lignin precursor, coniferyl alcohol, over sinapyl alcohol; its binding affinity for the former is about 2-fold higher than for the latter (see ). Fine tuning this variant by additional rounds of mutagenesis, together with positive (for coniferyl alcohol) and negative selection (for sinapyl alcohol) might engender enzymes specific for methylating the guaiacyl lignin precursor, with potential use in specifically disrupting the biosynthesis of condensed lignin.

Constitution and Biosynthesis of Lignin. Volume 2 …

Kinetic analysis showed that substituting Glu-165 with hydrophobic residues, like Phe, Ile, Val, Met, and Leu increased the binding affinity (Km) and the turnover (Vmax) for 4-O-methylation of monolignols (). The improvement of the catalytic activity of these mutant variants, particularly, of their binding affinity for monolignol generally correlates with the hydrophobicity of the substituted residues; the higher that was, the better the catalytic activity (). Structurally, those substitutions provided a more hydrophobic environment in the active site for binding aromatic substrates (). The variant E165F with a substitution showing the highest hydrophobicity yielded a catalytic ratio (Kcat/Km) for coniferyl alcohol at 287 −1 s−1, about 17-fold higher than that of IEMT wild-type. Substituting Glu-165 with the polar residue Ser also increased its binding affinity for coniferyl alcohol (); however, the catalytic activity of this variant did not rise proportionally. Presumably, this reflected the imposition by the side chain hydroxyl of Ser of an H-bond on the 4-O-methoxyl group of the product yielded, thus impairing its release. Enzyme activity was impaired by replacing Glu-165 with residues such as Tyr and Arg bearing a bulky side chain, or residue Pro that potentially entails severe changes in structural conformation ( and ). Particularly, changing Glu-165 to Arg totally abolished the activity for monolignol and isoeugenol (). These results suggest that the hydrophobic interaction and steric effect imposed by residue 165 on the phenyl ring of the bound compound in IEMT mutants play a crucial role in governing substrate binding and transmethylation proficiency.

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Constitution and Biosynthesis of Lignin Molecular …

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.

Constitution and biosynthesis of lignin (Molecular …

Directed evolution is a proven effective process for protein engineering and optimization (, ). Within the short time scale, a remarkable range of new enzymatic properties can be generated, based on creating different libraries and adopting different screening approaches. To create a novel enzyme exhibiting 4-OH methylation activity, preferentially for lignin monomeric precursors, primarily monolignols, i.e. a monolignol 4-O-methyltransferase (see C), we explored the structural basis of IEMT for its regioselective methylation and substrate discrimination by homology modeling. We identified seven putative “plasticity” amino acid residues in its active site that might contribute to the binding surface directly interacting with the accommodated phenolics. Thereafter, we established sets of enzyme-mutant libraries via site saturation and iterative saturation mutagenesis () primarily focusing on those identified plastic residues. Screening the activity of enzyme variants, we discovered that two evolutionarily plastic amino acid sites, Glu-165 and Thr-133, in the active site of IEMT dominate substrate specificity. Mutation at either site liberates the latent substrate flexibility of IEMT, enabling enzyme variants to accommodate lignin monomeric precursors, while retaining 4-O-methylation selectivity, i.e. the enzyme can methylate the para-hydroxyls of monolignols. The subsequent iterative saturation mutagenesis to optimize enzyme activity affirmed the additive effect of the beneficial mutations of both sites on the enzymes' catalytic efficiency; eventually, after three rounds of iterative mutagenesis, we obtained a triple mutant variant displaying more than a 70-fold increase in catalytic efficiency for the 4-O-methylation of monolignols compared with the parental enzyme. An in vitro polymerization assay confirmed that the 4-O-methoxy monolignol did not yield any type of oxidative coupling products, denoting that para-substitution of phenolics impairs the processes of dehydrogenation and oxidative coupling.

Biosynthesis and constitution of lignin

This similarity extended to other peroxidases involved in lignification, suggesting that a preferential structural organization of the substrate access channel for monolignol oxidation might exist in both angiosperms and gymnosperms.