High‐Throughput Colorimetric Detection and Quantification of Indoles and Pyrroloindoles for Enzymatic Activity Determination
Diana A. Amariei, Mona Haase, Moritz K. T. Klischan, Martin Wäscher, Jörg Pietruszka
Abstract
Abstract Indoles and pyrroloindoles are structural motifs present in many biologically active natural products. Multiple classes of enzymes catalyze the transformation of indoles into pyrroloindoles via group transfer followed by intramolecular cyclization, such as peroxydases, methyltransferases, and prenyltransferases. Due to the selective introduction of a stereogenic center, these enzymes receive increasing attention as catalytic tools for the production of pharmacologically relevant compounds. Two new colorimetric assays are described in this work, which allow for the quantification of such enzymatic reactions from the perspective of the substrate and the product. For the substrates, the indole assay is based on a modified version of the Ehrlich test, with the use of light as a driving force for color formation. The pyrroloindole assay uses cerium sulfate as a reagent for the colorimetric quantification of the enzymatic products. The assays are complementary and both were successfully utilized for enzymatic activity determination of a C3‐indole methyltransferase. They can facilitate high‐throughput screening of mutant libraries, offering support for the engineering of such enzymes, but can also be used as stand‐alone methods for the detection and quantification of natural products.