Pyrrolnitrin is a secondary metabolite derived from tryptophan and has strong antifungal activity. Recently we described four genes,prnABCD, from Pseudomonas fluorescens that encode the biosynthesis of pyrrolnitrin. In the work presented here, we describe the function of each prn gene product. The four genes encode proteins identical in size and serology to proteins present in wild-type Pseudomonas fluorescens, but absent from a mutant from which the entire prn gene region had been deleted. The prnA gene product catalyzes the chlorination of l-tryptophan to form 7-chloro-l-tryptophan. The prnB gene product catalyzes a ring rearrangement and decarboxylation to convert 7-chloro-l-tryptophan to monodechloroaminopyrrolnitrin. The prnC gene product chlorinates monodechloroaminopyrrolnitrin at the 3 position to form aminopyrrolnitrin. The prnD gene product catalyzes the oxidation of the amino group of aminopyrrolnitrin to a nitro group to form pyrrolnitrin. The organization of the prn genes in the operon is identical to the order of the reactions in the biosynthetic pathway.

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...

NADH statt Wasserstoffperoxid benötigen zwei neu identifizierte, substratspezifische und regioselektive Halogenasen. Beide wurden aus einem Pyrrolnitrin produzierenden Pseudomonas‐fluorescens‐Stamm isoliert; eine von ihnen katalysiert die Chlorierung von L‐Tryptophan zu 7‐Chlor‐L‐tryptophan 1.magnified image