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Enzymatic halogenation
08/2008 The incorporation of halogen atoms into organic compounds by living organisms is an enzyme-catalyzed reaction. The first halogenating enzyme was isolated from the fungus Caldariomyces fumago by L.P. Hager's group in 1966. This enzyme requires hydrogen peroxide and chloride, bromide or iodide for the halogenation of organic substrates suitable for electrophilic attack. This chloroperoxidase contains protoporphyrin IX as the prosthetic group. Hager and his group developed a spectrophotometric assay for the detection of haloperoxidases based on the chlorination and bromination of monochlorodimedone, a compound that has not been isolated from living organisms. Using this monochlorodimedone assay similar haloperoxidases were isolated from mammals, marine invertebrates, algae, and bacteria. In 1984, a novel type of haloperoxidase was detected by Vilter in the brown alga Ascophyllum nodosum. This bromoperoxidase is a non-haem haloperoxidase that requires vanadium for halogenating activity. Vanadium-dependent chloro- and bromoperoxidases were subsequently isolated from lichen, algae and fungi. A different type of non-haem haloperoxidase was isolated from bacteria in 1988 that neither contains metal ion nor requires any cofactor for haloperoxidase activity. The different types of haloperoxidases have in common that halogenation catalyzed by these enzymes lacks specificity. They show neither substrate specificity nor regioselectivity. This is explained by the reaction mechanism of haloperoxidases. Haem-containing and vanadium-dependent haloperoxidases directly produce hypohalous acid, whereas bacterial nonhaem haloperoxidases produce peracetic acid as an intermediate which can then oxidise halide ions. This halogenation catalyzed by peroxidases is actually a chemical halogenation reaction using hypohalous acid as halogenating agent. A different type of enzyme, a S-adenosyl methionine transferase, is involved in the formation of methyl halides. However, the involvement of S-adenosyl methionine transferase in halometabolite production seems to be restricted to the formation of single carbon metabolites. Biosynthetic studies on the formation of halogenated antibiotics produced by bacteria and comparison of the structures of halometabolites show that the halogenating enzymes involved in the formation of these metabolites must have substrate specificity and regioselectivity. However, specific halogenases can only be detected and isolated by using their natural substrate and not by using the monochlordimedone assay. Taking this into account, we were able to detect two halogenases in the bacterium Pseudomonas fluorescens that belong to a novel type of halogenating enzyme. The two novel halogenases do not require hydrogen peroxide; instead they need NADH. They are specific for their substrates, tryptophan and the phenyl pyrrole compound, monodechloroaminopyrrolnitrin respectively, and they catalyze the regiospecific halogenation of their substrates. Comparison of the corresponding genes show that they have no homology to each other. However, using the two genes as probes in hybridisation studies, it could be shown that similar enzymes are also present in a number of other bacteria producing different halometabolites. The novel NADH-dependent halogenases are more likely to be the halogenating enzymes involved in halometabolite formation than haloperoxidases, and they could be useful catalysts for the production of halogenated organic compounds without the formation of halogenated by-product. |
