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

Abstract

The indolocarbazole antitumor agent rebeccamycin is modified by chlorine atoms on each of two indole moieties of the aglycone scaffold. These halogens are incorporated during the initial step of its biosynthesis from conversion of L-Trp to 7-chlorotryptophan. Two genes in the biosynthetic cluster, rebF and rebH, are predicted to encode the flavin reductase and halogenase components of an FADH2-dependent halogenase, a class of enzymes involved in the biosynthesis of numerous halogenated natural products. Here, we report that, in the presence of O2, chloride ion, and L-Trp as cosubstrates, purified RebH displays robust regiospecific halogenating activity to generate 7-chlorotryptophan over at least 50 catalytic cycles. Halogenation by RebH required the addition of RebF, which catalyzes the NADH-dependent reduction of FAD to provide FADH2 for the halogenase. Maximal rates were achieved at a RebF/RebH ratio of 3:1. In air-saturated solutions, a k(cat) of 1.4 min(-1) was observed for the RebF/RebH system but increased at least 10-fold in low-pO2 conditions. RebH was also able to use bromide ions to generate monobrominated Trp. The demonstration of robust chlorinating activity by RebF/RebH sets up this system for the probing of mechanistic questions regarding this intriguing class of enzymes.

Fig. 1.

Chlorination of Trp to 7-chlorotryptophan as the initial step in the biosynthesis of rebeccamycin.

Fig. 2.

Formation of 7-chlorotryptophan by RebF/RebH. (A) Radio-HPLC traces of RebF/RebH reaction products derived from l-[¹⁴C]Trp showing time-dependent formation of 7-chlorotryptophan. Product formation requires O₂. When chloride ion was provided as [³⁶Cl]NaCl, l-Trp was converted to product containing ³⁶Cl label (trace at the bottom). (B) MALDI MS analysis of the [M + H]⁺ reaction product is consistent with formation of a monochlorinated Trp with the characteristic 3:1 M/M + 2 isotopic distribution of chlorine. (C) k_obs for RebH (6 μM) varies with RebF concentration (0–30 μM). Optimal activity for RebF/RebH is achieved at a RebF/RebH ratio of 3:1.

Fig. 3.

Bromination of l-Trp by RebF/RebH. Radio-HPLC of the RebF/RebH reaction supplemented with excess NaBr shows a new l-[¹⁴C]Trp-derived product with a retention time greater than that of 7-chlorotryptophan. The [M + H]⁺ compound mass and a 1:1 M/M + 2 mass ratio observed by MALDI-MS are indicative of a monobrominated Trp.

Fig. 4.

Proposed mechanism for halogenation by FADH₂-dependent halogenases. After formation of a FAD-OOH intermediate, the reactive chlorine is generated as a proposed FAD-O-Cl intermediate. Chlorination of l-Trp proceeds by attack of the aromatic π electrons on the FAD-O-Cl intermediate in a two-electron mechanism. Abstraction of a proton then generates the final product, 7-chlorotryptophan.