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

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Creating more space in the active site of the tryptophan 7‐halogenase PrnA by exchanging the large amino acid phenylalanine for the smaller alanine makes it possible for the substrate to bind in different orientations (see picture; yellow PrnA, blue PrnAF103A variant). This results in halogenation of the differently bound substrate in the 5‐position of the indole ring.

The biosynthetic gene cluster for the plant growth-regulating compound thienodolin was identified in and cloned from the producer organism Streptomyces albogriseolus MJ286-76F7. Sequence analysis of a 27 kb DNA region revealed the presence of 21 ORFs, 14 of which are involved in thienodolin biosynthesis. Three insertional inactivation mutants were generated in the sequenced region to analyze their involvement in thienodolin biosynthesis and to functionally characterize specific genes. The gene inactivation experiments together with enzyme assays with enzymes obtained by heterologous expression and feeding studies showed that the first step in thienodolin biosynthesis is catalyzed by a tryptophan 6-halogenase and that the last step is the formation of a carboxylic amide group catalyzed by an amidotransferase. The results led to a hypothetical model for thienodolin biosynthesis.

The ElmGT glycosyltransferase from S. olivaceus Tü2353 can transfer different sugars into the aglycon 8-DMTC. In addition to its natural sugar substrate L-rhamnose, ElmGT can transfer several L- and D-sugars and also a diolivosyl disaccharide into the aglycon 8-DMTC. ElmGT is an example of sugar flexible glycosyltransferase and can represent an important tool for combinatorial biosynthesis.