A flavin-dependent tryptophan 6-halogenase and its use in modification of pyrrolnitrin biosynthesis

Abstract

Regioselective halogenation of electron rich substrates is catalysed by flavin-dependent halogenases. Thienodolin produced by Streptomyces albogriseolus contains a chlorine atom in the 6-position of the indole ring system and is believed to be derived from tryptophan. Using the gene of the tryptophan 7-halogenase (PrnA) from the pyrrolnitrin biosynthetic gene cluster the gene for a tryptophan 6-halogenase was cloned, sequenced and heterologously overexpressed in Pseudomonas strains. In vitro activity of the purified enzyme could only be shown in a two-component enzyme system consisting of the halogenase, a flavin reductase, NADH, FAD and halide ions. The enzyme catalyses the regioselective chlorination and bromination of Land D-tryptophan. In its native form the enzyme is probably a homodimer with a relative molecular mass of the subunits of 63 600 (including the His-tag). Transformation of the pyrrolnitrin producer Pseudomonas chlororaphis ACN with a plasmid containing the tryptophan 6-halogenase gene lead to the formation of the new aminopyrrolnitrin derivative 3-(2?-amino-4?-chlorophenyl)pyrrole.

The Suzuki–Miyaura Cross-Coupling as a Versatile Tool for Peptide Diversification and Cyclization

“…Site-specific halogenases of tryptophan have also been developed for the preparation of halotryptophans. [35][36][37][38]76,77] Complex protein substrates that contain halogenated residues and which are not accessible via chemical synthesis could also be specifically obtained by means of auxotrophic strains or reassignment of stop codons [78,79]. Pioneering work by the group of P. G. Schultz expanded the genetic code via a unique tRNA/aminoacyl-tRNA synthetase pair, resulting in a solid protocol to insert 4-iodophenylalanine [80] and 4-boronophenylalanine [81].…”

Transformation with Tryptophan Halogenase Genes Leads to the Production of New Chlorinated Alkaloid Metabolites by a Medicinal Plant

“…[a] Prof. K. Co-expression of the tryptophan 5-or 6-halogenase genes pyrH or thal, [10] respectively, with the rebeccamycin or staurosporine biosynthetic genes in Streptomyces albus gave bisindoles halogenated at the 5-or 6-positions (Scheme 1; 6-9). [10] However, the downstream enzymes did not accept the halogenated derivatives as substrates.…”

Enzymatische Halogenierung von Tryptophan im Gramm‐Maßstab

“…[4][5][6][7] Aufgrund der Sandwich-artigen Bindung von l-Tryptophan im Inneren der aktiven Tasche der Halogenase wird selektiv die C7-Position für eine Chlorierung oder Bromierung zugänglich. [7,8] Der konservierte Lysinrest K79 wird dabei selektiv durch die hypohalogenige Säure oxidiert, wodurch die eigentliche halogenierende Spezies, ein langlebiges N-Halogenamin, gebildet wird.[9] Neben der Tryptophan-7-Halogenase RebH sind ebenso C5- [10] und C6-Tryptophan-Halogenasen [11] in der Literatur beschrieben. Enzym-katalysierte Reaktionen sind seit langem bekannt für ihre hohe Stereo-und Regioselektivität.…”