Eugenio P. Patallo

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.

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).

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.

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.

A Tryptophan 6-Halogenase and an Amidotransferase Are Involved in Thienodolin Biosynthesis

The biosynthetic gene cluster for the plant growth‐regulating compound thienodolin was identified in and cloned from the producer organism Streptomyces albogriseolus MJ286‐76F7. Sequence analysis of a 27 kb DNA region revealed the presence of 21 ORFs, 14 of which are involved in thienodolin biosynthesis. Three insertional inactivation mutants were generated in the sequenced region to analyze their involvement in thienodolin biosynthesis and to functionally characterize specific genes. The gene inactivation experiments together with enzyme assays with enzymes obtained by heterologous expression and feeding studies showed that the first step in thienodolin biosynthesis is catalyzed by a tryptophan 6‐halogenase and that the last step is the formation of a carboxylic amide group catalyzed by an amidotransferase. The results led to a hypothetical model for thienodolin biosynthesis.

Application and Modification of Flavin-Dependent Halogenases.

The application of flavin-dependent halogenases is hampered by their lack of stability under reaction conditions. However, first attempts to improve halogenase stability by error-prone PCR have resulted in mutants with higher temperature stability. To facilitate the screening for mutants with higher activity, a high-throughput assay was developed. Formation of cross-linked enzyme aggregates (CLEAs) of halogenases has increased halogenase lifetime by a factor of about 10, and CLEAs have been used to produce halogenated tryptophan in gram scale. Analyses of the substrate specificity of tryptophan halogenases have shown that they accept a much broader range of substrates than previously thought. The introduction of tryptophan halogenase genes into bacteria and plants led to the in vivo formation of peptides containing halogenated tryptophan or novel tryptophan-derived alkaloids, respectively. The halogen atoms in these compounds could be chemically exchanged against other substituents by cross-coupling reactions leading to novel compounds. Site-directed mutageneses have been used to modify the substrate specificity and the regioselectivity of flavin-dependent tryptophan halogenases. Since many flavin-dependent halogenases only accept protein-bound substrates, enzymatic and chemoenzymatic syntheses for protein-tethered substrates were developed, and the synthesized substrates were used in enzymatic halogenation reactions.

Characterization of the Aminotransferase ThdN from Thienodolin Biosynthesis in Streptomyces albogriseolus

In Streptomyces albogriseolus the indolethiophen alkaloid thienodolin is derived from tryptophan. The first step in thienodolin biosynthesis is the regioselective chlorination of tryptophan in the 6‐position of the indole ring. The second step is catalyzed by the aminotransferase ThdN. ThdN shows sequence homology (up to 69 % similarity) with known pyridoxal 5′‐phosphate‐dependent aminotransferases of the aspartate aminotransferase family from Gram‐positive bacteria. thdN was heterologously expressed in Pseudomonas fluorescens, and the enzyme was purified by nickel‐affinity chromatography. ThdN is a homodimeric enzyme with a mass of 90 600 kDa and catalyzes the conversion of l‐tryptophan and a number of chlorinated and brominated l‐tryptophans. The lowest KM values were found for 6‐bromo‐ and 6‐chlorotryptophan (40 and 66 μm, respectively). For l‐tryptophan it was 454 μm, which explains why thienodolin is the major product and dechlorothienodolin is only a minor component. The turnover number (kcat) for 7‐chlorotryptophan (128 min−1) was higher than that for the natural substrate 6‐chlorotryptophan (88 min−1).