A High‐Throughput Continuous Spectroscopic Assay to Measure the Activity of Natural Product Methyltransferases

Abstract

Natural product methyltransferases (NPMTs) represent an emerging class of enzymes that can be of great use for the structural and functional diversification of bioactive compounds, such as the strategic modification of C-, N-, O-and Smoieties. To assess the activity and the substrate scope of the ever-expanding repertoire of NPMTs, a simple, fast, and robust assay is needed. Here, we report a continuous spectroscopic assay, in which S-adenosyl-L-methionine-dependent methylation is linked to NADH oxidation through the coupled activities of S-adenosyl-L-homocysteine (SAH) deaminase and glutamate dehydrogenase. The assay is highly suitable for a high-throughput evaluation of small molecule methylation and for determining the catalytic parameters of NPMTs under conditions that remove the potent inhibition by SAH. Through the modular design, the assay can be extended to match the needs of different aspects of methyltransferase cascade reactions and respective applications.

Expanding the Substrate Scope of N‐ and O‐Methyltransferases from Plants for Chemoselective Alkylation**

“…PpCaOMT accepted all substrates containing a phenolic group (Figure 3). The acceptance of substrates 3, 4 and 6 by RgANMT as suggested previously [35] was confirmed; in addition, RgANMT Medicago sativa (Ms) CaOMT [31] 50.9 67.7 P28002.1 -+ + + *referring to the amino acid sequence of RgANMT. The protein from Citrus sinensis (KDO86634.1,) could not be used for activity assays due to solubility issues.…”

“…[34] In a recent paper describing an MT screening assay, initial insights into an extended substrate range towards other aniline substrates were reported. [35] To our knowledge, only one ANMT has been described to date, which makes a bioinformatic analysis of the underlying molecular differences challenging. In this study, we analysed a panel of putative N-and O-selective MTs regarding their substrate scopes.…”

Ein universeller, kontinuierlicher Assay für SAM‐abhängige Methyltransferasen

“…Simon‐Baram et al. haben kürzlich einen MT‐Assay veröffentlicht, der auf der Spaltung von SAH durch eine SAH‐Deaminase zur Bildung von S ‐Inosyl‐ l ‐Homocystein beruht, wobei Ammoniak entsteht, welches durch einen gekoppelten Glutamat‐Dehydrogenase‐Assay und der damit einhergehenden Absorptionsabnahme bei 340 nm, durch Umwandlung von NADH in NAD + , nachgewiesen werden kann (Schema 1B) [21] …”

“…B) Simon‐Baram et al. berichteten über die SAH‐Deaminase katalysierte Bildung von Ammoniak, die zu einer anschließenden Absorptionsabnahme aufgrund der Umwandlung von NADH in NAD + durch eine Glutamat‐Dehydrogenase führt [21] . C) Eine SAH‐Hydrolase bildet l ‐Homocystein und Adenosin.…”

ReACT (redox-activated chemical tagging) chemistry enables direct derivatization and fluorescence detection of S-adenosyl-l-homocysteine (SAH)

“…This can be challenging in the presence of other functionalities on the SAH scaffold such as hydroxyl, carboxyl and aromatic amino groups. In the past few years, a series of methods have been developed to measure the SAH concentration which can be divided into three major classes based on the detection mechanism: the chromatographic method coupled with a mass spectrometry or fluorescence detector, 10 the enzyme-coupled method which converts SAH to ATP, AMP, or other fluorescent/colorimetric products, 2 b ,11 and the molecular recognition method enabled by the SAH antibody or riboswitch. 12 Although great sensitivity and selectivity were achieved in various scenarios, these methods are accompanied by hurdles such as a sophisticated procedure, difficulty in accessing materials/facilities and potential ambiguities associated with a secondary protein.…”