NADH‐Dependent Halogenases Are More Likely To Be Involved in Halometaolite Biosynthesis Than Haloperoxidases

Abstract

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...

Biosynthesis of the dichloroacetyl component of chloramphenicol in Streptomyces venezuelae ISP5230: genes required for halogenation

“…In FAD-dependent hydroxylations the flavin is reduced by NAD(P)H and reacts with oxygen to form the 4(a)-flavin hydroperoxide; the hydroperoxide oxidizes the substrate to an epoxide, which is then opened by nucleophilic attack from a hydroxyl ion (Entsch et al, 1976;Gatti et al, 1994). A similar mechanism proposed for halogenasecatalysed reactions (Hohaus et al, 1997;Keller et al, 2000) is supported by recent evidence (van Pée, 2001) that the halogenase gene product requires FAD, NADPH, reductase activity and oxygen to function as a halogenating system. The epoxide generated by the flavin hydroperoxide undergoes nucleophilic attack by a halide ion, and specific removal of water gives the halogenated product.…”

“…Nevertheless, experiments with isotopically labelled potential precursors such as acetate, malonate and acetoacetate (Simonsen et al, 1978;Groß et al, 2002) have not firmly identified the substrate that, upon chlorination, provides the activated dichloroacetyl intermediate. Recently new avenues for exploring the mechanisms that introduce halogens into natural products have been opened by the discovery of FADH 2 -dependent halogenases (van Pée, 2001), and by demonstrations of their role in the biosynthesis of halogenated secondary metabolites (Hohaus et al, 1997;Kirner et al, 1998;NowakThompson et al, 1999;Keller et al, 2000;Puk et al, 2002). Halogenase genes have now been cloned from a variety of organisms: e.g.…”

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

“…Genetic manipulation in P. fluorescens BL915 has identified the intermediate products from each enzyme in the pyrrolnitrin pathway, leading to the current model for the biosynthetic pathway (4). 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).…”

Cloning, Sequencing, and Functional Analysis of an Iterative Type I Polyketide Synthase Gene Cluster for Biosynthesis of the Antitumor Chlorinated Polyenone Neocarzilin in “ Streptomyces carzinostaticus ”

“…Molecular genetic studies (9) identified the chl gene as being responsible for the chlorination of tetracycline in Streptomyces aureofaciens. Biochemical studies showed that a FADH 2 -dependent halogenase (PrnA) is involved in the biosynthesis of the tryptophan-derived halometabolite pyrrolnitrin in Pseudomonas fluorescens (20,27). Biosynthetic studies of NCZs could allow us to characterize a novel type of PKS for the polyenone skeleton and an as yet unknown halogenase involved in the biosynthesis of an aliphatic halometabolite.…”