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Literature summary extracted from

  • Cai, Y.; Bhuiya, M.; Shanklin, J.; Liu, C.
    Engineering a monolignol 4-O-methyltransferase with high selectivity for the condensed lignin precursor coniferyl alcohol (2015), J. Biol. Chem., 290, 26715-26724 .
    View publication on PubMedView publication on EuropePMC

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.1.1.146 purified recombinant MOMT5 and MOMT9 enzyme variants in complex with S-adenosyl-L-homocysteine (SAH), coniferyl alcohol, or sinapyl alcohol, hanging drop vapor diffusion method, crystallization from 22% (w/v) PEG 4000, 0.3 M Mg(NO3)2, pH 7.2, 1 mM DTT, 1 mM SAH, and 1 mM monolignols, X-ray diffraction structure determination and analysis at 1.60-1.73 A resolution, modeling Clarkia breweri

Protein Variants

EC Number Protein Variants Comment Organism
2.1.1.146 additional information a number of monolignol 4-O-methyltransferase variants (MOMTs) are evolved that can trans-methylate the para-hydroxyls of monolignols, which prevents the incorporation of modified precursors into the lignin polymer by disrupting oxidative radical formation. The engineered enzyme is biochemically active against both coniferyl and sinapyl alcohol with similar catalytic turnover rates (e.g. MOMT3 and 4). Therefore, the transgenic plants with overexpressed MOMT maintained similar monomer composition and lignin structure as the wild-type plants Clarkia breweri
2.1.1.146 T133L/E165I/F175I/F166W/H169F a promiscuous O-methyltransferase is incrementally evolved to constrain its substrate specificity to guaiacyl lignin precursors. The enzyme with nine amino acid substitutions is functionally specialized for selectively methylating the condensed lignin precursors. Specific remodeling the active site of a monolignol 4-O-methyltransferase creates an enzyme that specifically methylates the condensed guaiacyl lignin precursor coniferyl alcohol. The engineered promiscuous enzyme MOMT5 is built by remodelling of its substrate binding pocket by the addition of four substitutions, i.e. M26H, S30R, V33S, and T319M, yielding a mutant enzyme capable of discriminately etherifying the para-hydroxyl of coniferyl alcohol even in the presence of excess sinapyl alcohol. The engineered enzyme variant has a substantially reduced substrate binding pocket that imposes a clear steric hindrance thereby excluding bulkier lignin precursors Clarkia breweri

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.1.1.146 0.0114
-
 coniferyl alcohol pH 7.5, 30°C, recombinant enzyme mutant MOMT5 Clarkia breweri
2.1.1.146 0.0147
-
 sinapyl alcohol pH 7.5, 30°C, recombinant enzyme mutant MOMT5 Clarkia breweri
2.1.1.146 0.0161
-
 coniferyl alcohol pH 7.5, 30°C, recombinant enzyme mutant MOMT8 Clarkia breweri
2.1.1.146 0.0161
-
 coniferyl alcohol pH 7.5, 30°C, recombinant enzyme mutant MOMT9 Clarkia breweri
2.1.1.146 0.0263
-
 sinapyl alcohol pH 7.5, 30°C, recombinant enzyme mutant MOMT8 Clarkia breweri
2.1.1.146 0.0681
-
 sinapyl alcohol pH 7.5, 30°C, recombinant enzyme mutant MOMT4 Clarkia breweri
2.1.1.146 0.1095
-
 sinapyl alcohol pH 7.5, 30°C, recombinant enzyme mutant MOMT9 Clarkia breweri
2.1.1.146 0.1187
-
 sinapyl alcohol pH 7.5, 30°C, recombinant enzyme mutant MOMT3 Clarkia breweri
2.1.1.146 0.1926
-
 coniferyl alcohol pH 7.5, 30°C, recombinant enzyme mutant MOMT4 Clarkia breweri
2.1.1.146 0.1985
-
 coniferyl alcohol pH 7.5, 30°C, recombinant enzyme mutant MOMT3 Clarkia breweri

Organism

EC Number Organism UniProt Comment Textmining
2.1.1.146 Clarkia breweri O04385
-
-

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
2.1.1.146 0.0017
-
 enzyme variant MOMT4, pH 7.5, 30°C, substrate caffeyl alcohol Clarkia breweri
2.1.1.146 0.0025
-
 enzyme variant MOMT8, pH 7.5, 30°C, substrate coumaryl alcohol Clarkia breweri
2.1.1.146 0.0037
-
 enzyme variant MOMT9, pH 7.5, 30°C, substrate coumaryl alcohol Clarkia breweri
2.1.1.146 0.0055
-
 enzyme variant MOMT4, pH 7.5, 30°C, substrate coumaryl alcohol Clarkia breweri
2.1.1.146 0.0065
-
 enzyme variant MOMT5, pH 7.5, 30°C, substrate caffeyl alcohol Clarkia breweri
2.1.1.146 0.0077
-
 enzyme variant MOMT8, pH 7.5, 30°C, substrate sinapyl alcohol Clarkia breweri
2.1.1.146 0.0096
-
 enzyme variant MOMT5, pH 7.5, 30°C, substrate coumaryl alcohol Clarkia breweri
2.1.1.146 0.0098
-
 enzyme variant MOMT9, pH 7.5, 30°C, substrate sinapyl alcohol Clarkia breweri
2.1.1.146 0.0108
-
 enzyme variant MOMT8, pH 7.5, 30°C, substrate caffeyl alcohol Clarkia breweri
2.1.1.146 0.0129
-
 enzyme variant MOMT9, pH 7.5, 30°C, substrate caffeyl alcohol Clarkia breweri
2.1.1.146 0.0396
-
 enzyme variant MOMT5, pH 7.5, 30°C, substrate sinapyl alcohol Clarkia breweri
2.1.1.146 0.0514
-
 enzyme variant MOMT8, pH 7.5, 30°C, substrate coniferyl alcohol Clarkia breweri
2.1.1.146 0.0668
-
 enzyme variant MOMT4, pH 7.5, 30°C, substrate coniferyl alcohol Clarkia breweri
2.1.1.146 0.0683
-
 enzyme variant MOMT9, pH 7.5, 30°C, substrate coniferyl alcohol Clarkia breweri
2.1.1.146 0.0796
-
 enzyme variant MOMT4, pH 7.5, 30°C, substrate sinapyl alcohol Clarkia breweri
2.1.1.146 0.0806
-
 enzyme variant MOMT5, pH 7.5, 30°C, substrate coniferyl alcohol Clarkia breweri
2.1.1.146 0.1929
-
 enzyme variant MOMT3, pH 7.5, 30°C, substrate sinapyl alcohol Clarkia breweri
2.1.1.146 0.3463
-
 enzyme variant MOMT3, pH 7.5, 30°C, substrate coniferyl alcohol Clarkia breweri

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.1.1.146 additional information substrate specificities of engineered monolignol 4-O-methyltransferases, MOMT3, MOMT4, MOMT5, MOMT8, and MOMT9 Clarkia breweri ?
-
-
2.1.1.146 S-adenosyl-L-methionine + caffeyl alcohol
-
 Clarkia breweri S-adenosyl-L-homocysteine + 4-methoxycaffeyl alcohol
-
 ?
2.1.1.146 S-adenosyl-L-methionine + coniferyl alcohol
-
 Clarkia breweri S-adenosyl-L-homocysteine + 4-methoxyconiferyl alcohol
-
 ?
2.1.1.146 S-adenosyl-L-methionine + coumaryl alcohol
-
 Clarkia breweri S-adenosyl-L-homocysteine + 4-methoxycoumaryl alcohol
-
 ?
2.1.1.146 S-adenosyl-L-methionine + monolignol
-
 Clarkia breweri S-adenosyl-L-homocysteine + 4-O-methylmonolignol
-
 ?
2.1.1.146 S-adenosyl-L-methionine + sinapyl alcohol
-
 Clarkia breweri S-adenosyl-L-homocysteine + 4-methoxysinapyl alcohol
-
 ?

Synonyms

EC Number Synonyms Comment Organism
2.1.1.146 MOMT5
-
 Clarkia breweri
2.1.1.146 monolignol 4-O-methyltransferase
-
 Clarkia breweri

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.1.1.146 30
-
 assay at Clarkia breweri

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.1.1.146 0.006
-
 sinapyl alcohol pH 7.5, 30°C, recombinant enzyme mutant MOMT8 Clarkia breweri
2.1.1.146 0.017
-
 sinapyl alcohol pH 7.5, 30°C, recombinant enzyme mutant MOMT9 Clarkia breweri
2.1.1.146 0.033
-
 sinapyl alcohol pH 7.5, 30°C, recombinant enzyme mutant MOMT5 Clarkia breweri
2.1.1.146 0.049
-
 coniferyl alcohol pH 7.5, 30°C, recombinant enzyme mutant MOMT8 Clarkia breweri
2.1.1.146 0.063
-
 coniferyl alcohol pH 7.5, 30°C, recombinant enzyme mutant MOMT9 Clarkia breweri
2.1.1.146 0.069
-
 coniferyl alcohol pH 7.5, 30°C, recombinant enzyme mutant MOMT5 Clarkia breweri
2.1.1.146 0.173
-
 sinapyl alcohol pH 7.5, 30°C, recombinant enzyme mutant MOMT3 Clarkia breweri
2.1.1.146 0.247
-
 coniferyl alcohol pH 7.5, 30°C, recombinant enzyme mutant MOMT3 Clarkia breweri
2.1.1.146 0.271
-
 sinapyl alcohol pH 7.5, 30°C, recombinant enzyme mutant MOMT4 Clarkia breweri
2.1.1.146 0.528
-
 coniferyl alcohol pH 7.5, 30°C, recombinant enzyme mutant MOMT4 Clarkia breweri

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.1.1.146 7.5
-
 assay at Clarkia breweri

Cofactor

EC Number Cofactor Comment Organism Structure
2.1.1.146 S-adenosyl-L-methionine
-
 Clarkia breweri

General Information

EC Number General Information Comment Organism
2.1.1.146 additional information homology modeling and substrate docking analysis of enzyme mutant MOMT8, substrate binding in crystal structures of MOMTs, overview Clarkia breweri

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
2.1.1.146 0.16
-
 sinapyl alcohol pH 7.5, 30°C, recombinant enzyme mutant MOMT9 Clarkia breweri
2.1.1.146 0.23
-
 sinapyl alcohol pH 7.5, 30°C, recombinant enzyme mutant MOMT8 Clarkia breweri
2.1.1.146 1.24
-
 coniferyl alcohol pH 7.5, 30°C, recombinant enzyme mutant MOMT3 Clarkia breweri
2.1.1.146 1.46
-
 sinapyl alcohol pH 7.5, 30°C, recombinant enzyme mutant MOMT3 Clarkia breweri
2.1.1.146 2.25
-
 sinapyl alcohol pH 7.5, 30°C, recombinant enzyme mutant MOMT5 Clarkia breweri
2.1.1.146 2.74
-
 coniferyl alcohol pH 7.5, 30°C, recombinant enzyme mutant MOMT4 Clarkia breweri
2.1.1.146 3.04
-
 coniferyl alcohol pH 7.5, 30°C, recombinant enzyme mutant MOMT8 Clarkia breweri
2.1.1.146 3.91
-
 coniferyl alcohol pH 7.5, 30°C, recombinant enzyme mutant MOMT9 Clarkia breweri
2.1.1.146 3.98
-
 sinapyl alcohol pH 7.5, 30°C, recombinant enzyme mutant MOMT4 Clarkia breweri
2.1.1.146 6.05
-
 coniferyl alcohol pH 7.5, 30°C, recombinant enzyme mutant MOMT5 Clarkia breweri