Effects of Active-Site Modification and Quaternary Structure on the Regioselectivity of Catechol-O-Methyltransferase

Abstract

Catechol-O-methyltransferase (COMT), an important therapeutic target in the treatment of Parkinson's disease, is also being developed for biocatalytic processes, including vanillin production, although lack of regioselectivity has precluded its more widespread application. By using structural and mechanistic information, regiocomplementary COMT variants were engineered that deliver either meta- or para-methylated catechols. X-ray crystallography further revealed how the active-site residues and quaternary structure govern regioselectivity. Finally, analogues of AdoMet are accepted by the regiocomplementary COMT mutants and can be used to prepare alkylated catechols, including ethyl vanillin.

Keywords: biocatalysis; enzyme structure; methyltransferases; protein engineering; regioselectivity.

Figure 1

A) COMT methylation of substrates (1) to give meta (2) and para (3) products. B) mechanism of COMT‐catalyzed methylation of catechols using the cofactor S‐adenosyl‐l‐methionine (AdoMet). Coordination of Mg²⁺ by the catechol hydroxy groups lowers their pK _a values, thus aiding deprotonation of the hydroxy group closest to the AdoMet methyl group by K144. E199 appears to hydrogen bond with the other hydroxy group. The orientation of the R group in the active site determines whether the meta‐ or para‐hydroxy group is methylated.

Figure 2

Activities and regioselectivities of WT COMT and selected mutants. The conversion (blue bar) was calculated as the percentage of initial substrate converted after 20 min to both regioisomeric products combined. The regioisomeric excess (re; red bar) was calculated as the percentage excess of the meta regioisomer over the para regioisomer. Positive re values denote meta‐selectivity, whereas negative re values denote para‐selectivity. For all other mutants, see Tables S1–S4.

Figure 3

Crystal structures of COMT and proposed substrate binding modes for para‐ and meta‐methylation. Mg²⁺ (magenta sphere) is shown coordinated (dashed lines) to the catechol hydroxy groups. A) COMT mutant Y200L complexed with DNC, showing the Y200L mutation that causes the adjacent E199 residue to flip out of the active site and lose contact with the catechol. B) Crystal structure of WT COMT monomer complexed with DNC (PDB ID: 1VID), with E199 in close proximity to the R group of bound substrates. C) Overlay of the Y200L and WT structures, showing the altered positions of Y200L and E199 in the mutant (grey). D) Substrate orientation for meta‐methylation in Y200L. With E199 unable to hydrogen bond with the substrate aldehyde, the aldehyde group preferentially associates with the solvent, which positions the meta‐hydroxy group adjacent to the AdoMet sulphonium centre. E) Substrate orientation for para‐methylation in WT COMT, with the substrate aldehyde hydrogen bonding with E199, thereby orientating the catechol ring such that the para‐hydroxy group is presented to AdoMet.

Figure 4

A) Transfer of alkyl groups from AdoMet analogues to 1 a by using COMT. B) Comparison of conversions by Y200L COMT with AdoMet analogues after 20 min, and comparison of regioselectivity of Y200L versus WT COMT with AdoMet analogues. C) Enzymatic synthesis of ethyl vanillin (4 a) from ethionine.