New Insights into the Mechanism of Enzymatic Chlorination of Tryptophan

Abstract

Flavin-dependent halogenases have been shown to play a major role in biological halogenation reactions. For halogenating activity, flavin-dependent halogenases require reduced FAD, which is formed from FAD and NADH by a second enzyme, a flavin reductase. Although in a number of cases, a flavin reductase gene is present in the biosynthetic gene cluster of the halometabolites, it is unclear whether the corresponding flavin reductases interact directly with the halogenases. At least in a number of cases, flavin reductases from different bacterial strains can be used in combination with halogenases.1, 2-5 For the tryptophan 7-halogenase PrnA from Pseudomonas fluorescens BL915 which catalyzes the first step in pyrrolnitrin biosynthesis6 it could be shown that even chemically reduced FAD is used by the halogenase in the halogenation reaction.7 Based on the threedimensional structure of PrnA, it was postulated that flavin hydroperoxide is formed by the reaction of halogenase-bound reduced flavin with oxygen. This flavin hydroperoxide then reacts with chloride ion leading to the formation of hypochlorous acid, which is then guided along a tunnel about 10 Å long towards the substrate tryptophan (Figure 1). A lysine residue (K79) was suggested to hydrogen bond with hypochlorous acid and thus position it to react with tryptophan.8 Yeh et al. demonstrated chloramine formation by the reaction of HOCl with the ε-amino group of lysine,9 suggesting that chloramine rather than HOCl is the active agent. The importance of K79 is undisputed; exchange of K79 against an alanine residue leads to total loss of halogenating activity as demonstrated for PrnA8 and for the tryptophan 7-halogenase RebH from rebeccamycin biosynthesis.9 However, other factors must also be at work, since chlorination of tryptophan cannot be accomplished by chloramine (or HOCl) in solution.10, 11 Chloramine is a weaker halogenating agent than HOCl,12 and according to quantum mechanical calculations, N-chloramine formation reduces the electrophilicity ofthe chlorine species; in other words, the charge Q(Cl) is reduced to −0.07 compared to Q(Cl)=+0.017 in free HOCl.In the active site, glutamate 346 (E346) is positioned across the tunnel from K79, and the positioning of the substrate tryptophan is supported by a hydrogen bond between the NH group of the indole ring and the peptide bond oxygen between E346 and serine 347 (S347) (Figure 1). Evidence that E346 could be involved in the catalytic cycle was the observation that E346Q is two orders of magnitude less active.8 Whereas K79 is absolutely conserved in all the flavin-dependent halogenases known so far, E346 and S347 are conserved only in flavin-dependent tryptophan halogenases and they are not present in halogenases acting on substrates with a phenol or pyrrole ring. Dong et al. suggested that E346 is required for the ‡ Equal contribution

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).…”

Development of Halogenase Enzymes for Use in Synthesis

“…133 This electrophilic chlorine species is believed to be ultimately responsible for aromatic substitution of the substrate to generate the Wheland intermediate (23), which is then deprotonated by a conserved glutamate residue to afford chlorinated product (24, Figure 12). 114,131,[133][134][135] Positioning of this active site lysine relative to substrate is therefore believed to control which position of the substrate is halogenated.…”

“…114,123,127,132,134,135 chlorinated nonribosomal peptide kutzneride (27), an antifungal Hals (KtzQ and KtzR) and a KG dependent halogenase (KtzD), were required from kutzneride biosynthesis. [140][141][142] chlorotryptophan prior to Figure 14).…”

An Unusual Flavin-Dependent Halogenase from the Metagenome of the Marine Sponge Theonella swinhoei WA

“…[21][22][23] Unusually, KrmI appears to include a second, N-terminal domain-like region with distant homology to members of the ThiF enzyme family (Pfam family ThiF PF00899). Typically, these enzymes utilize ATP to activate carboxylic acids by adenylation.…”