Biochemical and Molecular Modeling Studies of the O-Methylation of Various Endogenous and Exogenous Catechol Substrates Catalyzed by Recombinant Human Soluble and Membrane-Bound Catechol-O-Methyltransferases

Abstract

Catechol-O-methyltransferase (COMT, EC 2.1.1.6) catalyzes the O-methylation of a wide array of catechol-containing substrates using s-adenosyl-L-methionine as the methyl donor. In the present study, we have cloned and expressed the human soluble and membrane-bound COMTs (S-COMT and MB-COMT, respectively) in Escherichia coli and have studied their biochemical characteristics for the O-methylation of representative classes of endogenous catechol substrates (catecholamines and catechol estrogens) as well as exogenous catechol substrates (bioflavonoids and tea catechins). Enzyme kinetic analyses showed that these two recombinant human COMTs are functionally active, with catalytic and kinetic properties nearly identical to those of crude or purified enzymes prepared from human tissues or cells. Kinetic parameters for the O-methylation of various substrates were characterized. In addition, computational modeling studies were conducted to better understand the molecular mechanisms for the different catalytic behaviors of human S- and MB-COMTs with respect to s-adenosyl-L-methionine, various substrates, and also the regioselectivity for the formation of mono-methyl ether products. Our modeling data showed that the binding energy values (Delta G) calculated for most substrates agreed well with the measured kinetic parameters. Also, our modeling data precisely predicted the regioselectivity for the O-methylation of these substrates at different hydroxyl groups, the predicted values matched nearly perfectly with the experimental data.

Methylation of Catechins and Procyanidins by Rat and Human Catechol-O-Methyltransferase: Metabolite Profiling and Molecular Modeling Studies

“…In contrast, the docking studies indicate that the orientation leading to 3Ј-O-methylation is energetically favorable. This conforms to the described binding mode of catecholic substrates to COMT (Palma et al, 2006;Bai et al, 2007) and our results in vitro, which show that 3Ј-O-methylation of catechins is clearly the major pathway. Therefore, we postulate that procyanidins may also be predominantly 3Ј-O-methylated by COMT.…”

Detoxication of Structurally Diverse Polycyclic Aromatic Hydrocarbon (PAH) o-Quinones by Human Recombinant Catechol-O-methyltransferase (COMT) via O-Methylation of PAH Catechols

“…SDS-polyacrylamide gel electrophoresis analysis gave a single band with an M r of 27,000 demonstrating that homogeneous recombinant enzyme had been obtained for our studies. The specific activities of COMT using pyrocatechol and epicatechin as substrates under standard enzyme assay conditions were 345 and 339 nmol of O-methylated catechol formed/min/mg, respectively, and these values were about 20-fold higher than those previously reported for the same substrates (35). We then proceeded to conduct PAH-catechol conjugation with this enzyme.…”

A catalytic triad – Lys-Asn-Asp – Is essential for the catalysis of the methyl transfer in plant cation-dependent O-methyltransferases

“…In both cases, the enzymes perform distinct regiospecific alkylation reactions of vicinal dihydroxy groups in the biosynthesis of natural products modulating their chemical reactivity and physiological properties (Vidgren et al, 1994;Hou et al, 2007). COMT in mammals essentially functions to inactivate potentially mutagenic catechols in the liver, and also the neurotransmitter L-DOPA in the brain (Männistö and Kaakkola, 1999;Zhu, 2002;Bai et al, 2007). In plants, one class of enzymes are referred to as caffeoyl CoA Omethyltransferases (CCoAOMTs), due to their most relevant physiological substrate, caffeoyl coenzyme A (Ye, 1997).…”