Karl-Heinz van Pée

Biological dehalogenation and halogenation reactions

A large number of halogenated compounds is produced by chemical synthesis. Some of these compounds are very toxic and cause enormous problems to human health and to the environment. Investigations on the degradation of halocompounds by microorganisms have led to the detection of various dehalogenating enzymes catalyzing the removal of halogen atoms under aerobic and anaerobic conditions involving different mechanisms. On the other hand, more than 3500 halocompounds are known to be produced biologically, some of them in great amounts. Until 1997, only haloperoxidases were thought to be responsible for incorporation of halogen atoms into organic compounds. However, recent investigations into the biosynthesis of halogenated metabolites by bacteria have shown that a novel type of halogenating enzymes, FADH(2)-dependent halogenases, are involved in biosyntheses of halogenated metabolites. In every gene cluster coding for the biosynthesis of a halogenated metabolite, isolated so far, one or several genes for FADH(2)-dependent halogenases have been identified.

Flavin-dependent halogenases involved in secondary metabolism in bacteria

The understanding of biological halogenation has increased during the last few years. While haloperoxidases were the only halogenating enzymes known until 1997, it is now clear that haloperoxidases are hardly, if at all, involved in biosynthesis of more complex halogenated compounds in microorganisms. A novel type of halogenating enzymes, flavin-dependent halogenases, has been identified as a major player in the introduction of chloride and bromide into activated organic molecules. Flavin-dependent halogenases require the activity of a flavin reductase for the production of reduced flavin, required by the actual halogenase. A number of flavin-dependent tryptophan halogenases have been investigated in some detail, and the first three-dimensional structure of a member of this enzyme subfamily, tryptophan 7-halogenase, has been elucidated. This structure suggests a mechanism involving the formation of hypohalous acid, which is used inside the enzyme for regioselective halogenation of the respective substrate. The introduction of halogen atoms into non-activated alkyl groups is catalysed by non-heme FeII α-ketoglutarate- and O2-dependent halogenases. Examples for the use of flavin-dependent halogenases for the formation of novel halogenated compounds in in vitro and in vivo reactions promise a bright future for the application of biological halogenation reactions.

Glycopeptide Biosynthesis in Amycolatopsis mediterranei DSM5908: Function of a Halogenase and a Haloperoxidase/Perhydrolase

Glycopeptides are important clinical emergency antibiotics consisting of a glycosylated and chlorinated heptapeptide backbone. The understanding of the biosynthesis is crucial for development of new glycopeptides. With balhimycin as a model system, this work focuses on the investigation of the putative halogenase gene (bhaA) and the putative haloperoxidase/perhydrolase gene (bhp) of the balhimycin biosynthesis gene cluster. An in-frame deletion mutant in the haloperoxidase/perhydrolase gene bhp (OP696) did not produce balhimycin. Feeding experiments revealed that bhp is involved in the biosynthesis of beta-hydroxytyrosine, a precursor of balhimycin. A bhaA in-frame deletion mutant (PH4) accumulated glycosylated but nonchlorinated balhimycin variants. The mutants indicated that only the halogenase BhaA is required for chlorination of balhimycin. Nonglycosylated and/or nonhalogenated metabolites can serve as starting points for combinatorial approaches for novel glycopeptides.

Structural Insights into Regioselectivity in the Enzymatic Chlorination of Tryptophan

The regioselectively controlled introduction of chlorine into organic molecules is an important biological and chemical process. This importance derives from the observation that many pharmaceutically active natural products contain a chlorine atom. Flavin-dependent halogenases are one of the principal enzyme families responsible for regioselective halogenation of natural products. Structural studies of two flavin-dependent tryptophan 7-halogenases (PrnA and RebH) have generated important insights into the chemical mechanism of halogenation by this enzyme family. These proteins comprise two modules: a flavin adenine dinucleotide (FAD)-binding module and a tryptophan-binding module. Although the 7-halogenase studies advance a hypothesis for regioselectivity, this has never been experimentally demonstrated. PyrH is a tryptophan 5-halogenase that catalyzes halogenation on tryptophan C5 position. We report the crystal structure of a tryptophan 5-halogenase (PyrH) bound to tryptophan and FAD. The FAD-binding module is essentially unchanged relative to PrnA (and RebH), and PyrH would appear to generate the same reactive species from Cl(-), O(2), and 1,5-dihydroflavin adenine dinucleotide. We report additional mutagenesis data that extend our mechanistic understanding of this process, in particular highlighting a strap region that regulates FAD binding, and may allow communication between the two modules. PyrH has a significantly different tryptophan-binding module. The data show that PyrH binds tryptophan and presents the C5 atom to the reactive chlorinating species, shielding other potential reactive sites. We have mutated residues identified by structural analysis as recognizing the tryptophan in order to confirm their role. This work establishes the method by which flavin-dependent tryptophan halogenases regioselectively control chlorine addition to tryptophan. This method would seem to be general across the superfamily.

New insights into the mechanism of enzymatic chlorination of tryptophan.

(Chemical Equation Presented) It takes two: Both a lysine and a glutamate residue in the active site of tryptophan halogenase are essential for its chlorination activity. A mechanism for the regioselective enzymatic chlorination of tryptophan involving both amino acids is suggested (see scheme).

A flavin-dependent tryptophan 6-halogenase and its use in modification of pyrrolnitrin biosynthesis

Regioselective halogenation of electron rich substrates is catalysed by flavin-dependent halogenases. Thienodolin produced by Streptomyces albogriseolus contains a chlorine atom in the 6-position of the indole ring system and is believed to be derived from tryptophan. Using the gene of the tryptophan 7-halogenase (PrnA) from the pyrrolnitrin biosynthetic gene cluster the gene for a tryptophan 6-halogenase was cloned, sequenced and heterologously overexpressed in Pseudomonas strains. In vitro activity of the purified enzyme could only be shown in a two-component enzyme system consisting of the halogenase, a flavin reductase, NADH, FAD and halide ions. The enzyme catalyses the regioselective chlorination and bromination of l- and d-tryptophan. In its native form the enzyme is probably a homodimer with a relative molecular mass of the subunits of 63 600 (including the His-tag). Transformation of the pyrrolnitrin producer Pseudomonas chlororaphis ACN with a plasmid containing the tryptophan 6-halogenase gene lead to the formation of the new aminopyrrolnitrin derivative 3-(2′-amino-4′-chlorophenyl) pyrrole.

Substrate Specificity and Regioselectivity of Tryptophan 7‐Halogenase from Pseudomonas fluorescens BL915

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.

Biological Halogenation has Moved far Beyond Haloperoxidases

Publisher Summary The first metabolite whose structural analysis showed that it contained a halogen atom was found in the marine eukaryote, Gorgonia cavolinii . This compound—3,5-diiodotyrosine—was later also isolated from the thyroid glands of mammals. Until 1961, only 29 halogenated organohalogen compounds had been isolated from living organisms. Although haloperoxidases have been isolated from organisms known to produce organohalogen compounds, it has never been demonstrated that these haloperoxidases are actually involved in the biosynthesis of these halometabolites; this raises the question whether haloperoxidases are actually the type of halogenating enzymes involved in the biosynthesis of secondary metabolites in microorganisms. The first halogenated metabolite identified in a microorganism was diploicidin. During the intensive search for antibiotics after the detection of penicillin, halogenated antibiotics such as chloramphenicol, 7-chlorotetracycline, vancomycin, and many others were isolated. Until now, more than 4000 organohalogens are known to be produced by living organisms. The number of producing organisms is comparable to the huge structural diversity of halometabolites. Organohalogen compounds have been isolated from bacteria, fungi, algae, lichen, higher plants, invertebrates, and vertebrates. However, halometabolites have not been detected in anaerobic organisms so far. Although many halometabolites show antibiotic or antitumor activity, their biological function for the producing organism is not known.

Detection of a new chloroperoxidase in Pseudomonas pyrrocinia

A new chloroperoxidase could be detected in Pseudomonas pyrrocinia ATCC 15 958, a bacterium that produces the antifungal antibiotic pyrrolnitrin. This enzyme was separated from a ferriprotoporphyrin IX containing bromoperoxidase which was also produced by this bacterium. The enzyme is capable of catalyzing the chorination of indole to 7‐chloroindole. This procaryotic chloroperoxidase requires the presence of H2O2 and can also brominate monochlorodimedone, but cannot catalyze its chlorination. This enzyme is the first chloroperoxidase described from procaryotic sources.

Changing the Regioselectivity of the Tryptophan 7‐Halogenase PrnA by Site‐Directed Mutagenesis

For many years, haloperoxidases were the only type of halogenating enzymes known. 2] Haloperoxidases (hemeand vanadium-containing) catalyze the formation of hypohalous acids, 4] which diffuse out of the active site and then react with substrate. Perhydrolases catalyze the formation of peracids, which react outside of the active site with halide ions to form hypohalous acids. In both cases the actual halogenation step initiated by haloperoxidases and perhydrolases is a nonenzymatic step consistent with the lack of substrate specificity and regioselectivity seen with these enzymes. The structures of many halogenated metabolites suggested that there are naturally occurring halogenating enzymes that have a high degree of substrate specificity and are capable of regioselective halogen incorporation. The halogenated indole (or tryptophan) derivatives serve as an elegant demonstration system since a series of derivatives can be isolated in which each individual position of the indole ring system has a halogen substituent. This clearly shows that halogenating enzymes with regioselectivity for each of the positions of the indole ring system must exist. The first halogenase found to catalyze the regioselective chlorination or bromination of tryptophan was the tryptophan 7-halogenase PrnA involved in pyrrolnitrin biosynthesis. PrnA was identified as a flavindependent halogenase requiring a flavin reductase as a second enzyme component. This flavin reductase produces FADH2 from flavin adenine dinucleotide (FAD) and reduced nicotinamide adenine dinucleotide (NADH; Scheme 1). FADH2 is bound by PrnA where it reacts with molecular oxygen to form a flavin hydroperoxide. A single chloride ion is bound close to the isoalloxazine ring of the FAD (Figure 1) and attacks the flavin hydroperoxide leading to the formation of hypochlorous acid. However, since the substrate tryptophan is bound about 10 away from the isoalloxazine ring, the hypochlorous acid is guided through a “tunnel” towards the substrate. In this process, a serine residue (S347), which is located halfway between the isoalloxazine ring and the substrate, seems to be involved. A lysine (K79) and a glutamate residue (E346) are located close to the substrate, and both are absolutely required for enzyme activity (Figure 2). 11] The lysine residue is suggested to react with the hypochlorous acid to form a chloramine as the halogenating intermediate. Flecks et al. suggested that a concerted interaction of hypochlorous acid with the lysine and the glutamate residue should increase the electrophilicity of the chlorine species and in addition ensure the correct positioning of the chlorine species for the regioselective incorporation of chlorine into the indole ring of tryptophan. Scheme 1. Reaction catalyzed by the two-component system of the flavin-dependent halogenases.