Altering the Regiospecificity of a CatecholO‐methyltransferase through Rational Design: Vanilloid vs. Isovanilloid Motifs in the B‐ring of Flavonoids

Abstract

Rational re-design of the substrate pocket of phenylpropanoidflavonoid O-methyltransferase (PFOMT) from Mesembryanthemum crystallinum, an enzyme that selectively methylates the 3'position (= meta-position) in catechol-moieties of flavonoids to guiacol-moieties, provided the basis for the generation of variants with opposite, i. e. 4'-(para-) regioselectivity and enhanced catalytic efficiency. A double variant (Y51R/N202W) identified through a newly developed colorimetric assay efficiently modified the para-position in flavanone and flavanonol substrates, providing access to the sweetener molecule hesperetin and other rare plant flavonoids having an isovanilloid motif.

Structural Characterization and Extended Substrate Scope Analysis of Two Mg2+‐Dependent O‐Methyltransferases from Bacteria**

“…Depending on the envisioned substrate, several active site residues of StrAOMT could be targeted by site‐directed mutagenesis. For dihydroxybenzoic acids and phenylpropanoids, manipulation of the putative carboxyl‐binding residues (such as R171) could force the opposite orientation of the substrate for enhanced para selectivity, as could the introduction of new positively charged residues instead of G213 or S184 [19,29] . The latter could also be mutated into a bulky residue to probe regioselectivity with larger substrates (flavonoids, coumarins, and anthraquinones).…”

“…Accordingly, engineering efforts to modulate the regioselectivity of COMTs have focused mainly on mutating the residues lining the catechol-binding pocket. A notable example is the Y51R mutation in PFOMT, which alone led to the production of a 1:1 mixture of methylated eriodyctiol products (as opposed to the exclusive meta methylation of the wild-type enzyme), while complete transition to para selectivity was achieved by an additional N202W mutation at the opposite site of the catechol pocket [19] . In that light, some of the promising candidates for site-directed mutagenesis include R171, I41 and D215 in StrAOMT or H160, A18 and K186 in DesAOMT.…”

“…For dihydroxybenzoic acids and phenylpropanoids, manipulation of the putative carboxyl-binding residues (such as R171) could force the opposite orientation of the substrate for enhanced para selectivity, as could the introduction of new positively charged residues instead of G213 or S184. [19,29] The latter could also be mutated into a bulky residue to probe regioselectivity with larger substrates (flavonoids, coumarins, and anthraquinones). For DesAOMT, however, more mechanistic insight is first needed before rational engineering can be attempted.…”

“…[10][11][12][13] Heterologously expressed OMTs have already been successfully integrated in engineered pathways towards high-value compounds like ferulic acid, [14] curcuminoids, [15] and vanillin, [16][17][18] with the latter resulting in the establishment of a commercial process. Additionally, a number of recent studies have focused on the fine-tuning of COMT regioselectivity, [12,19] but the substrate scope is mostly confined to plant phenylpropanoids and flavonoids. As O-methylation is one of the most common tailoring reactions in natural product biosynthesis alongside hydroxylation, glycosylation, and prenylation, promiscuous OMTs are a valuable asset in the toolkit for pathway engineering and diversity-oriented combinatorial biosynthesis.…”

Structural characterization and extended substrate scope analysis of two Mg2+-dependent O-methyltransferases from bacteria

“…Heterologously expressed OMTs have already been successfully integrated in the engineered pathways towards high-value compounds like ferulic acid [14] , curcuminoids [15] and vanillin [16][17][18] , with the latter resulting in the establishment of a commercial process. Additionally, a number of recent studies have focused on the fine-tuning of COMT regioselectivity [12,19] , but the substrate scope is mostly confined to plant phenylpropanoids and flavonoids. As O-methylation is one of the most common tailoring reactions in natural product biosynthesis alongside hydroxylation, glycosylation and prenylation, promiscuous OMTs are a valuable asset in the toolkit for pathway engineering and diversity-oriented combinatorial biosynthesis [20] .…”