Chlorination by a Long-Lived Intermediate in the Mechanism of Flavin-Dependent Halogenases^,

Abstract

The flavin-dependent halogenase RebH catalyzes the formation of 7-chlorotryptophan as the initial step in the biosynthesis of antitumor agent rebeccamycin. The reaction of FADH2, Cl-, and O2 in the active site generates the powerful oxidant HOCl, which was presumed to carry out the chlorination reaction. Herein, we demonstrate the formation of a long-lived chlorinating intermediate (t1/2 = 63 h at 4 degrees C) when RebH, FADH2, Cl-, and O2 react in the absence of substrate tryptophan. This intermediate remained on the enzyme after removal of FAD and transferred chlorine to tryptophan with kinetically competent rates. The identity of this intermediate is suggested by the X-ray crystal structure of RebH, which revealed an active site Lys79 located in a central position between flavin and tryptophan binding sites and just 4.1 A above C7 of tryptophan. The chlorinating species is proposed to be a Lys-epsilonNH-Cl (lysine chloramine) from reaction of enzyme-generated HOCl with the active site Lys79. This covalent enzyme chloramine likely plays a key role in directing regiospecific chlorination of substrate in this important class of biosynthetic enzymes.

Crystal structures and catalytic mechanism of cytochrome P450 StaP that produces the indolocarbazole skeleton

“…However, until now, the only crystal structure available was that of RebH which provides 7-chlorotryptophan, a precursor of rebeccamycin (11). Here, we report 2.4-Å and 1.9-Å resolution crystal structures of StaP in the presence and absence of the substrate CPA, respectively.…”

A GenoChemetic strategy for derivatization of the violacein natural product scaffold

“…All bisindole pathways use chromopyrrolic acid (CPA) as a common biosynthetic precursor, with the exception of violacein pathway which employs a unique VioE enzyme that forms the violacein 2-pyrrolinone core 26,27 . Although most bisindole pathways share the conserved ODCP genes that construct the indolocarbazole scaffold ( Figure S1B), some pathways such as the rebeccamycin pathway also contain a halogenase (RebH) and a flavin reductase (RebF) that are responsible for generating 7-chloro-L-tryptophan from L-tryptophan via a chloramine-lysine intermediate 28,29 . In addition, due to the functional similarity of .…”

The Ternary Complex of PrnB (the Second Enzyme in the Pyrrolnitrin Biosynthesis Pathway), Tryptophan, and Cyanide Yields New Mechanistic Insights into the Indolamine Dioxygenase Superfamily

“…The first enzyme, PrnA (tryptophan 7-halogenase), incorporates the chlorine into the substrate tryptophan (6). Structural and biochemical analyses of both tryptophan 7 and 5-halogenase (7)(8)(9)(10) have established a novel chemical mechanism of hypohalous acid formation at the flavin cofactor, followed by N-chlorolysine formation (9) and finally regioselective halogenation of tryptophan (controlled by orientation of substrate) (10,11). PrnB converts 7-Cl-tryptophan into monodechloroamino-pyrrolnitrin, although the chirality of the PrnB substrate has remained unclear (4).…”