Influence of Substrate Binding Residues on the Substrate Scope and Regioselectivity of a Plant O‐Methyltransferase against Flavonoids

Abstract

Methylation of free hydroxyl groups is an important modification for flavonoids. It not only greatly increases absorption and oral bioavailability of flavonoids, but also brings new biological activities. Flavonoid methylation is usually achieved by a specific group of plant O‐methyltransferases (OMTs) which typically exhibit high substrate specificity. Here we investigated the effect of several residues in the binding pocket of the Clarkia breweri isoeugenol OMT on the substrate scope and regioselectivity against flavonoids. The mutation T133M, identified as reported in our previous publication, increased the activity of the enzyme against several flavonoids, namely eriodictyol, naringenin, luteolin, quercetin and even the isoflavonoid genistein, while a reduced set of amino acids at positions 322 and 326 affected both, the activity and the regioselectivity of the methyltranferase. On the basis of this work, methylated flavonoids that are rare in nature were produced in high purity.

Directed Evolution of a Halide Methyltransferase Enables Biocatalytic Synthesis of Diverse SAM Analogs

“…The T133M-Y326L variant of isoeugenol O-MT (IeOMT) and the V140T AtHMT catalyzed regioselective mono-ethylation of luteolin to 3'-O-ethylluteolin (6) with 41 % conversion. [15] Similarly, human catechol O-MT (COMT) in cascade with V140T AtHMT catalyzed regioselective mono-allylation of 3,4dihydroxybenzaldehyde to produce 4-allyloxy-3hydroxybenzaldehyde (8) with 48 % conversion (Table S5). [16] The identities of the products were confirmed by 1 H and 13 C NMR spectroscopy (Figures S12-S17).…”

“…Therefore, we used the V140T At HMT for in vitro regeneration of SAE, SAP, and SAA, from catalytic amounts of SAH, in one‐pot biocatalytic alkylation cascades (Figure 3 b). The T133M–Y326L variant of isoeugenol O ‐MT (IeOMT) and the V140T At HMT catalyzed regioselective mono‐ethylation of luteolin to 3′‐ O ‐ethylluteolin ( 6 ) with 41 % conversion [15] . Similarly, human catechol O ‐MT (COMT) in cascade with V140T At HMT catalyzed regioselective mono‐allylation of 3,4‐dihydroxybenzaldehyde to produce 4‐allyloxy‐3‐hydroxybenzaldehyde ( 8 ) with 48 % conversion (Table S5) [16] .…”

Evolution of DIMBOA-Glc O-Methyltransferases from Flavonoid O-Methyltransferases in the Grasses

“…This indicates a similar orientation and binding mode for DIMBOA-Glc and flavonoid substrates in the active sites of these enzymes. Previous publications showed that the substrate preference, regiospecificity, and overall activity of FOMTs can be altered by a few amino acid mutations in the substrate binding pocket [ 16 , 43 , 44 ]. Using homology modeling and amino acid sequence comparisons, we identified nine putative active site residues that differ between flavonoid 5- and DIMBOA-Glc 4-OMTs ( Figure 3 , Figures S4 and S5 ).…”

Die gerichtete Evolution einer Halogenid‐Methyltransferase erlaubt die biokatalytische Synthese diverser SAM‐Analoga

“…Methyltransferase 6) mit 41 % Umsatz. [15] Ebenso katalysierte die humane Catechol-O-MT (COMT) in einer Kaskade mit V140T-AtHMT die regioselektive Monoallylierung von 3,4-Dihydroxybenzaldehyd, um 4-Allyloxy-3-hydroxybenzaldehyd (8) bei 48 % Umsatz zu bilden (Tabelle S5). [16] Die Identität der Produkte wurde über 1 H-und 13 Wir haben gezeigt, dass das Spektrum biokatalytischer Alkylierungen rasch durch Ausnutzung der Promiskuität einer optimierten Halogenid-Methyltransferase erweitert werden kann.…”

“…Daher verwendeten wir die V140T‐ At HMT für die In‐vitro‐Regenerierung von SAE, SAP und SAA mit katalytischen Mengen von SAH in Eintopf‐biokatalytischen Alkylierungskaskaden (Abbildung 3 b). Die T133M‐Y326L‐Variante der Isoeugenol O ‐MT (IeOMT) und die V140T‐ At HMT katalysierten die regioselektive Monoethylierung von Luteolin zu 3′‐ O ‐Ethylluteolin ( 6 ) mit 41 % Umsatz [15] . Ebenso katalysierte die humane Catechol‐ O ‐MT (COMT) in einer Kaskade mit V140T‐ At HMT die regioselektive Monoallylierung von 3,4‐Dihydroxybenzaldehyd, um 4‐Allyloxy‐3‐hydroxybenzaldehyd ( 8 ) bei 48 % Umsatz zu bilden (Tabelle S5) [16] .…”