The detection of chloroperoxidase from the fungus Caldariomycesfumagofll and the development of a simple spectrophotometric assay12] for the detection of halogenating enzymes based on the synthetic compound monochlorodimedone (1) as organic substrate resulted in the subsequent isolation of a number of haloperoxidases from different organisms. All H3C CH3these enzymes produce hypohalogenic acid, which is the actual halogenating agent. Thus, halogenation catalyzed by haloperoxidases lacks substrate and regiospecificity.13 -41 However, investigations of the biosynthetic pathways of different halometabolites have shown that biological halogenation must be specific.[3. 51 Furthermore, the formation of fluorinated metabolites by haloperoxidases is difficult to explain, as fluoride cannot be oxidized in the haloperoxidase reaction.[61 Recently, genetic investigations showed that haloperoxidasetype enzymes are definitely not involved in the biosynthesis of chlorotetracycline and pyrr~lnitrin.~' -These results raise some interesting questions. What other type of halogenating enzymes could exist, and how can they be detected? It had always been assumed that the enzyme oxidizes the halide ion and that the oxidized halide reacts with the organic substrate. However, why couldn't the enzyme first react with the organic substrate in a way that would make it suitable for nucleophilic attack by the halide ion itself?Apparently all groups working on enzymatic halogenation have ignored the fact that, if they were looking for specific enzymes, they should use the natural substrates for these enzymes and not a substrate like 1. One reason that this approach was ignored is the lack of knowledge about the structure of these substrates. Thus, prior to the use of a "natural" substrate it had to be established that this compound actually is halogenated in vivo.Tryptophan (2) would be such a substrate, if the chlorination of 2 to 7-chlorotryptophan (3) is the first step in the biosynthesis of the antifungal antibiotic pyrrolnitrin (6, Scheme 1) .I9] To check this hypothesis, the growth medium of a mutant of Pseudomonasfluorescens blocked in the second step of pyrrolnitrin biosynthesis was analyzed. The isolated 3 was identified as the L-isomer by empioying D-and L-amino acid oxidases. Thus, chlorination of the L-isomer of 2 was identified as the first step in pyrrolnitrin biosynthesis by P.fluorescens, and this strain therefore must contain an enzyme that catalyzes the specific chlorination of the L-isomer of 2 to the L-isomer of 3. A second chlorination occurs later, where monodechloroaminopyrrolnitrin (4) is chlorinated to aminopyrrolnitrin (5, Scheme I) .f91Using the L-isomer of 2 as the substrate and a Pseudomonasfluorescens mutant that lacked all chromosomal pyrrolnitrin-biosynthesis genes but harbored the gene for the first step on a plasmid, we searched for tryptophan halogenase activity by means of an HPLC assay. As we did not know what kind of cofactor, if any, would be needed, a number of different cofactors were tested. The chlo...

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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.

The prnABCD gene cluster from Pseudomonas fluorescens encodes the biosynthetic pathway for pyrrolnitrin, a secondary metabolite derived from tryptophan which has strong anti-fungal activity. We used the prn genes from P. fluorescens strain BL915 as a probe to clone and sequence homologous genes from three other Pseudomonas strains, Burkholderia cepacia and Myxococcus fulvus. With the exception of the prnA gene from M. fulvus59% similar among the strains, indicating that the biochemical pathway for pyrrolnitrin biosynthesis is highly conserved. The prnA gene from M. fulvus is about 45% similar to prnA from the other strains and contains regions which are highly conserved among all six strains.

A non-haem chloroperoxidase was isolated from the enteric bacterium Serratia marcescens. The enzyme was purified to homogeneity by heat treatment, ammonium sulfate precipitation, ion exchange chromatography, gel filtration and dye-ligand affinity chromatography. Native chloroperoxidase has a molecular mass of 58 kDa and consists of two identical subunits of 29 kDa. Whereas chloroperoxidase catalyses only the bromination of monochlorodimedone, indole is chlorinated by this enzyme. Chloroperoxidase also catalyses the oxidation of amino to nitro groups. The enzyme is thermostable and does not lose any activity when incubated at 65 degrees C for 2 h. Comparison of the first 15 amino-terminal amino acids showed a sequence identity of 80% to the chloroperoxidases from Streptomyces lividans and Pseudomonas pyrrocinia. However, no precipitation band was obtained in the Ouchterlony agar diffusion assay with antibodies raised against the chloroperoxidases from Pseudomonas pyrrocinia and Streptomyces aureofaciens Tü24.