Substrate Specificity and Regioselectivity of Tryptophan 7-Halogenase fromPseudomonas fluorescensBL915

Abstract

Tryptophan 7‐halogenase which is involved in pyrrolnitrin biosynthesis is the first halogenating enzyme to be isolated that has substrate specificity and regioselectivity. This FADH2‐dependent halogenase catalyzes the chlorination of its natural substrate tryptophan exclusively at the 7‐position, a position at which direct chemical chlorination is not possible. Other substrates such as N‐Ω‐methyltryptamine, 5‐methyltryptamine, 5‐methylindole, 3‐methylindole, or indole‐3‐acetonitrile are also chlorinated by the enzyme, whereas compounds like 1‐methyltryptophan, indole‐3‐carboxylic acid, indole‐3‐acetic acid, or indole are not accepted as substrates. In addition, phenylpyrrole derivatives are also chlorinated by the enzyme. However, in contrast to tryptophan, the tryptophan and indole derivatives are chlorinated at positions 2 or/and 3 of the indole ring system and not at the 7‐position. Chlorination of the phenylpyrrole derivatives also proceeds without regioselectivity and a mixture of mono‐ and dichlorinated products is obtained.

Robust in vitro activity of RebF and RebH, a two-component reductase/halogenase, generating 7-chlorotryptophan during rebeccamycin biosynthesis

“…Despite the many representatives of this halogenase class and its role in the biosynthesis of medicinally important halogenated aromatic natural products, only a few of these catalysts have been characterized (9)(10)(11). PrnA, the Trp-7-halogenase involved in the formation of pyrrolnitrin, is foremost among FADH 2 -dependent halogenases that have been described (9,10).…”

Regioselective Arene Halogenation using the FAD‐Dependent Halogenase RebH

“…The large structural differences between these substrates and 2-amino Several notable differences between the reactivity RebH and that reported for PrnA deserve comment given the significant homology of both the complete sequences (55%) and the active sites (vide infra) of these two enzymes. First, PrnA was reported to have no activity on indole, gramine, or 1-methyl-L-tryptophan, [11] whereas RebH halogenated each of these substrates. The simplest explanation for this difference is that low conversion using PrnA precluded identification of the halogenated products.…”

“…[14] As part of one such effort, O'Connor and coworkers demonstrated that tryptamine is accepted by RebH and is halogenated at the 7-position, [15] which contrasts with the preference for halogenation of the 2-position of this substrate by PrnA. [11] Inspired by these examples, we set out to explore the synthetic utility of RebH in preparative halogenation reactions. We focused on this enzyme and its cognate flavin reductase, RebF, due to reports of their expression in E. coli, [13] which facilitates both enzyme production and genetic manipulation.…”

“…Characterization of their activity in vitro has typically involved analytical scale reactions of tryptophan, [9] but van Pée and coworkers also explored the substrate scope of the tryptophan-7-halogenase PrnA expressed in Pseudomonas fluorescens (Scheme 2). [11] A variety of substituted indoles were accepted by the enzyme, but halogenation invariably occurred at the electronically most activated indole 2-position for all substrates except tryptophan. These same researchers were able to alter the selectivity of PrnA to produce 5-chloro-and 5-bromotryptophan by mutating a single active site residue, but 7-halogenation remained the dominant reaction.…”

“…Second, PrnA was reported to catalyze 2-halogenation of both 5-and N--methyltryptamine, [11] while RebH catalyzes 6-and 7-halogenation of these substrates, respectively. Crystal structures for both of these enzymes bound to FAD, chloride, and tryptophan are available.…”