Microbial biosynthesis of halometabolites

Halometabolites are compounds that are commonly found in nature and they are produced by many different organisms. Whereas bromometabolites can mainly be found in the marine environment, chlorometabolites are predominately produced by terrestrial organisms; iodo- and fluorocompounds are only produced infrequently. The halogen atoms are incorporated into organic compounds by enzyme-catalyzed reactions with halide ions as the halogen source. For over 40 years haloperoxidases were thought to be responsible for the incorporation of halogen atoms into organic molecules. However, haloperoxidases lack substrate specificity and regioselectivity, and the connection of haloperoxidases with the in vivo formation of halometabolites has never been demonstrated. Recently, molecular genetic investigations showed that, at least in bacteria, a different class of halogenases is involved in halometabolite formation. These halogenases were found to require FADH2, which can be produced from FAD and NADH by unspecific flavin reductases. In addition to FADH2, oxygen and halide ions (chloride and bromide) are necessary for the halogenation reaction. The FADH2-dependent halogenases show substrate specificity and regioselectivity, and their genes have been detected in many halometabolite-producing bacteria, suggesting that this type of halogenating enzymes constitutes the major source for halometabolite formation in bacteria and possibly also in other organisms.     

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⁻, O₂, 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.     

Enzymatic halogenation catalyzed via a catalytic triad and by oxidoreductases

During the search for haloperoxidases in bacteria we detected a type of enzymes that catalyzed the peroxide-dependent halogenation of organic substrates. However, in contrast to already known haloperoxidases, these enzymes do not contain a prosthetic group or metal ions nor any other cofactor. Biochemical and molecular genetic studies revealed that they contain a catalytic triad consisting of a serine, a histidine, and an aspartate. The reaction they catalyze is actually the perhydrolysis of an acetic acid serine ester leading to the formation of peracetic acid. As a strong oxidizing agent the enzymatically formed peracetic acid can oxidize halide ions, resulting in the formation of hypohalous acid which then acts as the actual halogenating agent. Since hypohalous acid is also formed by the heme- and vanadium-containing haloperoxidases, enzymatic halogenation catalyzed by haloperoxidases and perhydrolases in general lacks substrate specificity and regioselectivity. However, detailed studies on the biosynthesis of several halometabolites led to the detection of a novel type of halogenases. These enzymes consist of a two-component system and require NADH and FAD for activity. Whereas the gene for one of the components is part of the biosynthetic cluster of the halometabolite, the second component is an enzyme which is also present in bacteria from which no halometabolites have ever been isolated, like Escherichia coli. In contrast to haloperoxidases and perhydrolases the newly detected NADH/FAD-dependent halogenases are substrate-specific and regioselective and might provide ideal tools for specific halogenation reactions.     

Catalytic mechanisms, basic roles, and biotechnological and environmental significance of halogenating enzymes

The understanding of enzymatic incorporation of halogen atoms into organic molecules has increased during the last few years. Two novel types of halogenating enzymes, flavindependent halogenases and α-ketoglutarate-dependent halogenases, are now known to play a significant role in enzyme-catalyzed halogenation. The recent advances on the halogenating enzymes RebH, SyrB2, and CytC3 have suggested some new mechanisms for enzymatic halogenations. This review concentrates on the occurrence, catalytic mechanisms, and biotechnological applications of the halogenating enzymes that are currently known.     

Molecular mechanisms of enzyme-catalysed halogenation

Since their discovery, halogenated metabolites have been somewhat of a biological peculiarity and it is only now that we are beginning to realize the full extent of their medicinal value. With the exception of the well characterized haloperoxidases, most of the biosynthetic enzymes and mechanisms responsible for the halogenations have remained elusive. The crystal structures of two functionally diverse halogenases have been recently solved, providing us with new and exciting mechanistic detail. This new insight has the potential to be used both in the development of biomimetic halogenation catalysts and in engineering halogenases, and related enzymes, to halogenate new substrates. Interestingly, these new structures also illustrate how the evolution of these enzymes mirrors that of the monooxygenases, where the cofactor is selected for its ability to generate a powerful oxygenating species. In this highlight article we will examine the proposed catalytic mechanisms of the halogenases and how these relate to their structures. In addition, we will consider how this chemistry might be harnessed and developed to produce novel enzymatic activity.     

色氨酸卤化酶研究进展

能催化卤代反应的卤化酶,因其具有高效、选择性好、反应条件温和的特点受到广泛关注。其中色氨酸卤化酶是研究最多的一类酶,它的特点是可以有选择性地卤化色氨酸,主要包括氯化和溴化。而卤代化合物具有多种生物活性,在医药、化工等领域有着广泛的应用。本文主要介绍了色氨酸卤化酶的来源和种类,酶学性质及其异源表达的研究现状;重点阐述了其结构和功能之间的相互关系及催化机理;并对色氨酸卤化酶的应用以及未来发展方向进行了展望,以期为色氨酸卤化酶的开发应用提供参考。     

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.     

Chloramphenicol Biosynthesis: The Structure of CmlS, a Flavin-Dependent Halogenase Showing a Covalent Flavin-Aspartate Bond

Chloramphenicol is a halogenated natural product bearing an unusual dichloroacetyl moiety that is critical for its antibiotic activity. The operon for chloramphenicol biosynthesis in Streptomyces venezuelae encodes the chloramphenicol halogenase CmlS, which belongs to the large and diverse family of flavin-dependent halogenases (FDH’s). CmlS was previously shown to be essential for the formation of the dichloroacetyl group. Here we report the X-ray crystal structure of CmlS determined at 2.2 Å resolution, revealing a flavin monooxygenase domain shared by all FDHs, but also a unique ‘winged-helix’ C-terminal domain that creates a T-shaped tunnel leading to the halogenation active site. Intriguingly, the C-terminal tail of this domain blocks access to the halogenation active site, suggesting a structurally dynamic role during catalysis. The halogenation active site is notably nonpolar and shares nearly identical residues with Chondromyces crocatus tyrosyl halogenase (CndH), including the conserved Lys (K71) that forms the reactive chloramine intermediate. The exception is Y350, which could be used to stabilize enolate formation during substrate halogenation. The strictly conserved residue E44, located near the isoalloxazine ring of the bound flavin adenine dinucleotide (FAD) cofactor, is optimally positioned to function as a remote general acid, through a water-mediated proton relay, which could accelerate the reaction of the chloramine intermediate during substrate halogenation, or the oxidation of chloride by the FAD(C4α)-OOH intermediate. Strikingly, the 8α carbon of the FAD cofactor is observed to be covalently attached to D277 of CmlS, a residue that is highly conserved in the FDH family. In addition to representing a new type of flavin modification, this has intriguing implications for the mechanism of FDHs. Based on the crystal structure and in analogy to known halogenases, we propose a reaction mechanism for CmlS.     

Bacterial haloperoxidases and their role in secondary metabolism

Bacteria produce a large number of different halogenated secondary metabolites. Haloperoxidases are believed to be the enzymes responsible for the halogenation reaction. Two classes of haloperoxidases, heme and nonheme, were isolated from different bacteria and their role in the biosynthesis of halogenated secondary metabolites was investigated. Two genes of bacterial haloperoxidases were cloned and can now be used to produce large quantities of the enzymes.     

Optimisation of halogenase enzyme activity by application of a genetic algorithm

A genetic algorithm (GA) was applied for the optimisation of an enzyme assay composition respectively the enzyme activity of a recombinantly produced FADH(2)-dependent halogenating enzyme. The examined enzyme belongs to the class of halogenases and is capable to halogenate tryptophan regioselective in position 5. Therefore, the expressed trp-5-halogenase can be an interesting tool in the manufacturing of serotonin precursors. The application of stochastic search strategies (e.g. GAs) is well suited for fast determination of the global optimum in multidimensional search spaces, where statistical approaches or even the popular classical one-factor-at-a-time method often failures by misleading to local optima. The concentrations of six different medium components were optimised and the maximum yield of the halogenated tryptophan could be increased from 3.5 up to 65%.     

Exploring the Chemistry and Biology of Vanadium-dependent Haloperoxidases

Nature has developed an exquisite array of methods to introduce halogen atoms into organic compounds. Most of these enzymes are oxidative and require either hydrogen peroxide or molecular oxygen as a cosubstrate to generate a reactive halogen atom for catalysis. Vanadium-dependent haloperoxidases contain a vanadate prosthetic group and utilize hydrogen peroxide to oxidize a halide ion into a reactive electrophilic intermediate. These metalloenzymes have a large distribution in nature, where they are present in macroalgae, fungi, and bacteria, but have been exclusively characterized in eukaryotes. In this minireview, we highlight the chemistry and biology of vanadium-dependent haloperoxidases from fungi and marine algae and the emergence of new bacterial members that extend the biological function of these poorly understood halogenating enzymes.     

Binding of FAD and tryptophan to the tryptophan 6‐halogenase Thal is negatively coupled

Flavin‐dependent halogenases require reduced flavin adenine dinucleotide (FADH₂), O₂, and halide salts to halogenate their substrates. We describe the crystal structures of the tryptophan 6‐halogenase Thal in complex with FAD or with both tryptophan and FAD. If tryptophan and FAD were soaked simultaneously, both ligands showed impaired binding and in some cases only the adenosine monophosphate or the adenosine moiety of FAD was resolved, suggesting that tryptophan binding increases the mobility mainly of the flavin mononucleotide moiety. This confirms a negative cooperativity between the binding of substrate and cofactor that was previously described for other tryptophan halogenases. Binding of substrate to tryptophan halogenases reduces the affinity for the oxidized cofactor FAD presumably to facilitate the regeneration of FADH₂ by flavin reductases.     

A Fivefold Parallelized Biosynthetic Process Secures Chlorination of Armillaria mellea (Honey Mushroom) Toxins

The basidiomycetous tree pathogen Armillaria mellea (honey mushroom) produces a large variety of structurally related antibiotically active and phytotoxic natural products, referred to as the melleolides. During their biosynthesis, some members of the melleolide family of compounds undergo monochlorination of the aromatic moiety, whose biochemical and genetic basis was not known previously. This first study on basidiomycete halogenases presents the biochemical in vitro characterization of five flavin-dependent A. mellea enzymes (ArmH1 to ArmH5) that were heterologously produced in Escherichia coli. We demonstrate that all five enzymes transfer a single chlorine atom to the melleolide backbone. A 5-fold, secured biosynthetic step during natural product assembly is unprecedented. Typically, flavin-dependent halogenases are categorized into enzymes acting on free compounds as opposed to those requiring a carrier-protein-bound acceptor substrate. The enzymes characterized in this study clearly turned over free substrates. Phylogenetic clades of halogenases suggest that all fungal enzymes share an ancestor and reflect a clear divergence between ascomycetes and basidiomycetes.     

Halogenation and Oxidation Reactions with Haloperoxidases

Haloperoxidases are enzymes which catalyze the incorporation of halogen atoms into organic molecules. They are found throughout nature, playing a major role in the defence system of many organisms. Their reaction mechanisms as well as their use as catalysts for halogenation and oxidation reactions on laboratory and industrial scales are discussed. Up to now, selective halogenation reactions have only been reported for the chloroperoxidase from Pseudomonas pyrrocinia. The usefulness of the other enzymes is based on their ability to produce hypohalous acid (HOX) in a controllable way, allowing the smooth (yet nonselective) halogenation of electron-rich substrates. On the other hand, it has been shown recently that some haloperoxidases can stereoselectively convert sulfides and alkenes into their corresponding homochiral oxides. Therefore, these enzymes will undoubtedly gain importance in the near future.     

Heme-thiolate haloperoxidases: versatile biocatalysts with biotechnological and environmental significance

Heme-thiolate haloperoxidases are undoubtedly the most versatile biocatalysts of the hemeprotein family and share catalytic properties with at least three further classes of heme-containing oxidoreductases, namely, classic plant and fungal peroxidases, cytochrome P450 monooxygenases, and catalases. For a long time, only one enzyme of this type—the chloroperoxidase (CPO) of the ascomycete Caldariomyces fumago—has been known. The enzyme is commercially available as a fine chemical and catalyzes the unspecific chlorination, bromination, and iodation (but no fluorination) of a variety of electrophilic organic substrates via hypohalous acid as actual halogenating agent. In the absence of halide, CPO resembles cytochrome P450s and epoxidizes and hydroxylates activated substrates such as organic sulfides and olefins; aromatic rings, however, are not susceptible to CPO-catalyzed oxygen-transfer. Recently, a second fungal haloperoxidase of the heme-thiolate type has been discovered in the agaric mushroom Agrocybe aegerita. The UV–Vis adsorption spectrum of the isolated enzyme shows little similarity to that of CPO but is almost identical to a resting-state P450. The Agrocybe aegerita peroxidase (AaP) has strong brominating as well as weak chlorinating and iodating activities, and catalyzes both benzylic and aromatic hydroxylations (e.g., of toluene and naphthalene). AaP and related fungal peroxidases could become promising biocatalysts in biotechnological applications because they seemingly fill the gap between CPO and P450 enzymes and act as “self-sufficient” peroxygenases. From the environmental point of view, the existence of a halogenating mushroom enzyme is interesting because it could be linked to the multitude of halogenated compounds known from these organisms.     

Halogenation and Oxidation Reactions with Haloperoxidases

Haloperoxidases are enzymes which catalyze the incorporation of halogen atoms into organic molecules. They are found throughout nature, playing a major role in the defence system of many organisms. Their reaction mechanisms as well as their use as catalysts for halogenation and oxidation reactions on laboratory and industrial scales are discussed. Up to now, selective halogenation reactions have only been reported for the chloroperoxidase from Pseudomonas pyrrocinia. The usefulness of the other enzymes is based on their ability to produce hypohalous acid (HOX) in a controllable way, allowing the smooth (yet nonselective) halogenation of electron-rich substrates. On the other hand, it has been shown recently that some haloperoxidases can stereoselectively convert sulfides and alkenes into their corresponding homochiral oxides. Therefore, these enzymes will undoubtedly gain importance in the near future.     

Halogenase engineering and its utility in medicinal chemistry

Halogenation is commonly used in medicinal chemistry to improve the potency of pharmaceutical leads. While synthetic methods for halogenation present selectivity and reactivity challenges, halogenases have evolved over time to perform selective reactions under benign conditions. The optimization of halogenation biocatalysts has utilized enzyme evolution and structure-based engineering alongside biotransformation in a variety of systems to generate stable site-selective variants. The recent improvements in halogenase-catalyzed reactions has demonstrated the utility of these biocatalysts for industrial purposes, and their ability to achieve a broad substrate scope implies a synthetic tractability with increasing relevance in medicinal chemistry.     

Structure and Action of the Myxobacterial Chondrochloren Halogenase CndH: A New Variant of FAD-dependent Halogenases

The crystal structure of the FAD-dependent chondrochloren halogenase CndH has been established at 2.1 Å resolution. The enzyme contains the characteristic FAD-binding scaffold of the glutathione reductase superfamily. Except for its C-terminal domain, the chainfold of CndH is virtually identical with those of FAD-dependent aromatic hydroxylases. When compared to the structurally known FAD-dependent halogenases PrnA and RebH, CndH lacks a 45 residue segment near position 100 and deviates in the C-terminal domain. Both variations are near the active center and appear to reflect substrate differences. Whereas PrnA and RebH modify free tryptophan, CndH halogenates the tyrosyl group of a chondrochloren precursor that is most likely bound to a carrier protein. In contrast to PrnA and RebH, which enclose their small substrate completely, CndH has a large non-polar surface patch that may accommodate the putative carrier. Apart from the substrate binding site, the active center of CndH corresponds to those of PrnA and RebH. At the halogenation site, CndH has the characteristic lysine (Lys76) but lacks the required base Glu346 (PrnA). This base may be supplied by a residue of its C-terminal domain or by the carrier. These differences were corroborated by an overall sequence comparison between the known FAD-dependent halogenases, which revealed a split into a PrnA-RebH group and a CndH group. The two functionally established members of the CndH group use carrier-bound substrates, whereas three members of PrnA-RebH group are known to accept a free amino acid. Given the structural and functional distinction, we classify CndH as a new variant B of the FAD-dependent halogenases, adding a new feature to the structurally established variant A enzymes PrnA and RebH.     

Riboflavin deficiency is associated with selective preservation of critical flavoenzyme-dependent metabolic pathways.

Riboflavin is a water soluble vitamin that serves as a precursor of flavin mononucleotide and flavin adenine dinucleotide. These two compounds are coenzymes in a variety of electron transfer reactions that occur in energy producing, biosynthetic, detoxifying and electron scavenging pathways. When an organism is confronted with inadequate dietary riboflavin, characteristic changes occur in the cellular distribution of the various flavin fractions as well as in the activities of flavin-dependent enzymes. These changes suggest a specific hierarchic response to riboflavin deficiency, e.g. the core electron transfer chain required for ATP synthesis is preserved while the enzymes required for the first step of fatty acid beta-oxidation are diminished. The mechanisms by which the specific changes in enzyme activity are mediated have not been completely identified, but appear to result from a combination of diminished access of normal or near normal levels of apoenzyme to coenzyme and diminished abundance of apoenzyme. The changes in apoenzyme content potentially result from alterations in either protein stability or gene expression. The response to riboflavin deficiency of several key enzyme systems and the pathways affected will be discussed and a hierarchic order by which specific enzyme activities are preserved while others are decreased will be proposed. The current understanding of the molecular mechanisms by which these changes are mediated will be discussed.     

Flavogenomics--a genomic and structural view of flavin-dependent proteins

Riboflavin (vitamin B(2)) serves as the precursor for FMN and FAD in almost all organisms that utilize the redox-active isoalloxazine ring system as a coenzyme in enzymatic reactions. The role of flavin, however, is not limited to redox processes, as ～ 10% of flavin-dependent enzymes catalyze nonredox reactions. Moreover, the flavin cofactor is also widely used as a signaling and sensing molecule in biological processes such as phototropism and nitrogen fixation. Here, we present a study of 374 flavin-dependent proteins analyzed with regard to their function, structure and distribution among 22 archaeal, eubacterial, protozoan and eukaryotic genomes. More than 90% of flavin-dependent enzymes are oxidoreductases, and the remaining enzymes are classified as transferases (4.3%), lyases (2.9%), isomerases (1.4%) and ligases (0.4%). The majority of enzymes utilize FAD (75%) rather than FMN (25%), and bind the cofactor noncovalently (90%). High-resolution structures are available for about half of the flavoproteins. FAD-containing proteins predominantly bind the cofactor in a Rossmann fold (～ 50%), whereas FMN-containing proteins preferably adopt a (βα)(8)-(TIM)-barrel-like or flavodoxin-like fold. The number of genes encoding flavin-dependent proteins varies greatly in the genomes analyzed, and covers a range from ～ 0.1% to 3.5% of the predicted genes. It appears that some species depend heavily on flavin-dependent oxidoreductases for degradation or biosynthesis, whereas others have minimized their flavoprotein arsenal. An understanding of 'flavin-intensive' lifestyles, such as in the human pathogen Mycobacterium tuberculosis, may result in valuable new intervention strategies that target either riboflavin biosynthesis or uptake.