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Volume 84 Issue 21 | pp. 12-18
Issue Date: May 22, 2006

Cover Story

Nature's X-Factors

Enzymes responsible for installing halogen atoms in natural products with exquisite selectivity reveal their catalytic logic
Department: Science & Technology
News Channels: Biological SCENE
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Last Resort
Nature uses a mononuclear iron (orange) enzyme for its toughest halogenation jobs: halogenating unactivated organic molecules. The enzyme's α-ketoglutarate cofactor is shown in gray; chlorine, green ball; and water, aqua ball.
Credit: Courtesy of Leah Blasiak
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Last Resort
Nature uses a mononuclear iron (orange) enzyme for its toughest halogenation jobs: halogenating unactivated organic molecules. The enzyme's α-ketoglutarate cofactor is shown in gray; chlorine, green ball; and water, aqua ball.
Credit: Courtesy of Leah Blasiak

Before you read any further, think of a few organohalogen compounds commonly found in the environment. If you're like most people, your first thought will be man-made pollutants such as chlorofluorocarbons and dioxins. But in fact, nature's inventory of organohalogens is dizzyingly complex: Some 4,500 halogenated natural products, many with promising medicinal properties, have been identified to date. "And that list grows by more than 100 new natural organohalogens per year," says chemist and organohalogen aficionado Gordon W. Gribble of Dartmouth College.

The ways in which bacteria, fungi, marine organisms, animals, and even humans produce these halogenated molecules have long intrigued chemists. A variety of chemical halogenation methods are available to the synthetic chemist, but none are so exquisitely specific as the enzymes nature uses to install halogen atoms, notes Christopher T. Walsh, a professor of biological chemistry and molecular pharmacology at Harvard Medical School. He and other scientists are working to reveal the structural and mechanistic secrets of nature's arsenal of halogenation enzymes "in the hopes that we might learn the catalytic rules of halogenation and perhaps use them to halogenate other substrates," he says.

Walsh notes that nature turns to halogenation to fine-tune a natural product's biological properties. "Enzymatic incorporation of halogens during natural product assembly alters physical properties," including electronic and steric effects that can determine the affinity and selectivity of the molecule's interaction with its biological target. The chlorinated antibiotic vancomycin is a perfect example. One of its two chlorines enhances the compound's binding affinity to its cell-wall target, whereas the other is responsible for the compound's binding selectivity for the same target, he explains.

Likewise, halogenation has remained a popular tool for tweaking a drug candidate's biological properties for nearly a century, notes medicinal chemist Paul S. Anderson, now retired after a 40-year career in the pharmaceutical industry. Medicinal chemists used halogenation chemistry to modify early drug leads in projects such as the discovery of sulfa drugs in the early 20th century, he notes. Today, up to 20% of drugs on the market and up to a quarter of those in the development pipeline are halogenated, mostly with fluorine, according to David O'Hagan, a chemistry professor at the University of St. Andrews, in Scotland.

Anderson notes that fluorine and chlorine are more commonly used in pharmaceuticals than are bromine or iodine "for metabolic, chemical stability, and cost-of-goods reasons." Most halogenated natural products, though, contain either chlorine or bromine, and in some cases, both. Only a few dozen fluorinated and iodinated metabolites have been characterized.

Nature installs halogen atoms on all sorts of organic scaffolds at all types of carbon centers, including aromatic and heterocyclic rings, olefinic sites, and even aliphatic carbons. Recent work has shown that the type of catalyst nature selects for each of these jobs depends on the chemical challenge a given halogen installation presents, Walsh notes.

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The Right Tool
The type of catalyst that nature selects for a particular halogen installation varies. A vanadium haloperoxidase is involved in snyderol biosynthesis (blue), a heme-iron haloperoxidase in tetraiodothyronine (red) biosynthesis, and a flavin-dependent halogenase in chlortetracycline (yellow) biosynthesis. Each halogenase acts on an electron-rich substrate. For unactivated substrates, nature uses mononuclear iron halogenases like the one involved in biosynthesizing barbamide (green).
Credit: Courtesy of Chris Walsh and Ellen Yeh
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The Right Tool
The type of catalyst that nature selects for a particular halogen installation varies. A vanadium haloperoxidase is involved in snyderol biosynthesis (blue), a heme-iron haloperoxidase in tetraiodothyronine (red) biosynthesis, and a flavin-dependent halogenase in chlortetracycline (yellow) biosynthesis. Each halogenase acts on an electron-rich substrate. For unactivated substrates, nature uses mononuclear iron halogenases like the one involved in biosynthesizing barbamide (green).
Credit: Courtesy of Chris Walsh and Ellen Yeh

Nearly all halogenase enzymes—chlorinases, brominases, and iodinases—elect to oxidize abundant halide ions (X-) to electrophilic X+ ions or radical X??? species that can attack a substrate in the active site.

The one enzyme known to carry out fluorination, however, marches to the beat of a different drummer. The energy required to oxidize fluoride ion is prohibitively high, says O'Hagan, whose lab studies a prototypical fluorinase enzyme involved in the bacterial biosynthesis of fluoroacetate and the amino acid fluorothreonine. Instead, the fluorinase enzyme relies on a constellation of amino acid side chains in the active site to strip fluoride ion of its protective cloak of water, he explains. The naked fluoride ion then undergoes a simple SN2 substitution reaction with a substrate to generate a C-F bond, he says.

Walsh suggests that fluoride's incompatibility with nature's standard oxidative halogenation strategies "may be the major reason why natural organofluorine compounds are rare in biology." In the past year, he and other enzymologists have triggered a radical transformation of our understanding of biological halogenation.

For many years, the halogenation of natural products seemed to be limited to a single family of enzymes known as the haloperoxidases, then the only known halogenases. These enzymes can have one of two cofactors: heme iron or vanadium. Whatever metal cofactor is at their center, haloperoxidases use hydrogen peroxide to generate a metal-bound hypohalite ion. The electrophilic X+ ion that is generated halogenates the substrate positioned in the active site.

Natural products made with heme iron haloperoxidase include the human endocrine hormone tetraiodothyronine. A vanadium enzyme has been implicated in the biosynthesis of snyderol and other marine natural products.

In each of these natural products, the positioning of the haloperoxidase's substrate in the active site determines the site of halogenation, notes chemist Alison Butler of the University of California, Santa Barbara. For example, in snyderol biosynthesis, bromination by vanadium haloperoxidase occurs at just one face of a terminal olefin, leading to diasteroselective cyclization to give the brominated snyderol, she says. But whatever their cofactor, she points out, haloperoxidases can halogenate only double bonds of electron-rich substrates.

Of course, in nature there's always more than one way to skin a cat. In the mid-1990s, a Japanese group isolated the halogenase responsible for installing chlorine in the antibiotic chlortetracycline. Its gene sequence showed no similarity to those of the haloperoxidases, confirming many scientists' suspicions that haloperoxidases were not the only halogenases existing. Intrigued, biochemist Karl-Heinz van Pée of the Technical University of Dresden, in Germany, took a closer look at the biosynthetic pathway for the chlorinated antifungal natural product pyrrolnitrin. The halogenase he found—PrnA, which installs chlorine at the chemically inaccessible C-7 position of tryptophan—is similar to that involved in chlortetracycline biosynthesis. Neither enzyme contains a metal, only flavin adenine dinucleotide, an organic redox cofactor.

Speculation abounded over what sort of halogenating agent such a flavin-dependent enzyme might use to regioselectively halogenate its substrate. Last fall, van Pée, in collaboration with structural enzymologist James H. Naismith and postdoc Changjiang Dong of St. Andrews University, nailed down the halogenating agent, and it surprised everyone, even van Pée.

Harkening back to the haloperoxidases, PrnA's flavin cofactor uses O2 and chloride ion to generate none other than hypochlorous acid (HOCl). The team's structure revealed a tunnel that guides the HOCl from the flavin to the substrate 10Å. away (Science 2005, 309, 2216). "The first thing HOCl sees when it comes out of that tunnel is the 7 position" of tryptophan, van Pée says.

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Guided Missile
A 10-Å-long tunnel guides HOCl produced by the flavin cofactor (bottom, black) of tryptophan-7-halogenase (ribbon) to its tryptophan substrate (top, black). The two chlorines captured in the crystal structure are shown in green.
Credit: Courtesy of Jim Naismith
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Guided Missile
A 10-Å-long tunnel guides HOCl produced by the flavin cofactor (bottom, black) of tryptophan-7-halogenase (ribbon) to its tryptophan substrate (top, black). The two chlorines captured in the crystal structure are shown in green.
Credit: Courtesy of Jim Naismith

A nearby, positively charged lysine side chain helps activate the HOCl for electrophilic aromatic substitution, he adds. He and Naismith hope to learn more about how the enzyme positions its substrate to halogenate in the right spot by studying the structures of two close relatives that chlorinate tryptophan at the 5 and 6 positions, respectively.

Many more such flavin-dependent halogenases, each of which halogenates different substrates, have yet to be discovered, van Pée explains. But as is the case for the haloperoxidases, flavin-dependent halogenases' reliance on X+ as a halide donor prevents them from halogenating anything but electron-rich substrates, he admits.

The chemical limitations of haloperoxidases and flavin-dependent halogenases initially had chemists like William H. Gerwick of UC San Diego and Scripps Institution of Oceanography scratching their heads. Gerwick and others had turned up a treasure trove of wild-looking natural products with halogen groups that "couldn't possibly be made with known enzymes." In each metabolite, the carbon center to be halogenated is chemically unreactive and electron poor, he explains.

"We suspected radical reactions would be required to introduce these halogen atoms," Gerwick says. He found evidence for his hunch in isotope-feeding experiments with a Caribbean cyanobacterium that produces barbamide, a potent molluscicide containing a trichloromethyl group. The experiments suggested that the trichloromethyl group came from a trichloroleucine precursor. His chemical intuition led him to suggest that a radical halogenation strategy was at work, an idea that at the time was considered "heretical," Gerwick says.

But he turned out to be right. In 2002, Gerwick and David H. Sherman of the University of Michigan isolated the cluster of genes responsible for barbamide biosynthesis and found no haloperoxidases and no flavoproteins. What they did find was a pair of enzymes that resembled a type of metalloenzyme that generates a high-energy radical intermediate to hydroxylate unactivated substrates. "We knew right away that these must be the halogenases we were looking for," Sherman says.

And indeed they were. Soon, Walsh's postdoc Frederic Vaillancourt pinpointed a handful of these suspected halogenases in biosynthetic gene clusters for a variety of natural products, including the leaf toxin coronatine and the antibiotic syringomycin. Vaillancourt and Walsh then demonstrated that, like their hydroxylase counterparts, these halogenases are α-ketoglutarate dependent and contain a mononuclear iron center capable of generating a highly reactive oxo-iron species during catalysis (Nature 2005, 436, 1191).

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Cryptic Chlorination
Chlorination is a key step in the biosynthesis of the leaf toxin coronatine, although you wouldn't know it by looking at the natural product's structure. A mononuclear iron halogenase catalyzesthe key chlorination (red) of a tethered L-allo-isoleucine (left), paving the way for another enzyme to catalyze intramolecularγ-elimination and cyclopropane formation.
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Cryptic Chlorination
Chlorination is a key step in the biosynthesis of the leaf toxin coronatine, although you wouldn't know it by looking at the natural product's structure. A mononuclear iron halogenase catalyzesthe key chlorination (red) of a tethered L-allo-isoleucine (left), paving the way for another enzyme to catalyze intramolecularγ-elimination and cyclopropane formation.

To shed light on how this oxo-iron species carries out the necessary oxidation of chloride ion to a Cl species that can halogenate an unactivated alkane, Vaillancourt and Walsh teamed up with Massachusetts Institute of Technology crystallographer Catherine L. Drennan and grad student Leah C. Blasiak to structurally characterize SyrB2, which catalyzes the chlorination of threonine during syringomycin biosynthesis (Nature 2006, 440, 368).

They found that both α-ketoglutarate and a chloride ion are coordinated to the iron center. Walsh suggests that the highly reactive oxo-iron species generated by SyrB2 first abstracts a hydrogen from its alkane substrate, which can then hook up with the metal-bound Cl species to give the chlorinated product.

Walsh finds the parallels between nature's hydroxylation and halogenation strategies "fascinating." Nature uses flavin-dependent hydroxylases on electron-rich substrates, he notes, but "rolls out the big guns" of mononuclear iron enzymes to hydroxylate unreactive alkanes. "The halogenation logic is remarkably similar," he says. In fact, the active sites of mononuclear iron hydroxylases and halogenases differ by just a single amino acid. Walsh's lab has now teamed up with seasoned hydroxylase enzymologists J. Martin Bollinger Jr. and Carsten Krebs of Pennsylvania State University to probe what determines whether these enzymes act as halogenases or hydroxylases.

The halogenases may prove to be more than just fascinating curiosities to enzymologists, Walsh argues. They could open the door to new, useful halogenation chemistry. Already, a number of nonnatural organohalogens have been produced by feeding organisms alternative or radiolabeled halides. O'Hagan is working with researchers at GlaxoSmithKline to use his fluorinase enzyme to make 18F-labeled compounds that could be used as contrast agents for positron emission tomography.

In addition, vanadium bromoperoxidase has been used to synthesize eight-membered cyclic bromoethers in the lab, a reaction "that can be tough to do chemically," Butler tells C&EN.

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Access Problem
In SyrB2, as in many other halogenases, the substrate to be halogenated is delivered to a deeply buried active site (shown at the base of the tunnel in the cutaway at left) on the end of a long flexible arm (right). This delivery constraint will likely present a challenge to those wishing to use these enzymes in combinatorial biosynthesis of novel organohalogens.
Credit: Courtesy of Leah Blasiak
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Access Problem
In SyrB2, as in many other halogenases, the substrate to be halogenated is delivered to a deeply buried active site (shown at the base of the tunnel in the cutaway at left) on the end of a long flexible arm (right). This delivery constraint will likely present a challenge to those wishing to use these enzymes in combinatorial biosynthesis of novel organohalogens.
Credit: Courtesy of Leah Blasiak

Researchers also hope to make novel organohalogens by mixing and matching the components of biosynthetic gene clusters encoding halogenated natural products. Flavin-dependent tryptophan halogenases have already proven useful in such combinatorial biosynthesis strategies. Microbiologist José A. Salas of the University of Oviedo, in Spain, and synthetic chemist Jurgen Rohr of the University of Kentucky have produced by fermentation a variety of rebeccamycin derivatives with chlorine atoms in novel positions. They did so by substituting 5-tryptophan and 6-tryptophan halogenase genes for the 7-tryptophan halogenase gene of the rebeccamycin gene cluster (Proc. Natl. Acad. Sci. USA 2005, 102, 461).

O'Hagan and Jonathan B. (Joe) Spencer of the University of Cambridge have similar hopes for fluorinase. O'Hagan tells C&EN that Spencer's lab has finally isolated the cluster of genes responsible for fluorometabolite biosynthesis, including the gene that allows a fluoroacetate-producing organism to resist being killed by this toxic metabolite. With this fluorinase gene cluster in hand, they hope to insert it into other organisms to produce novel organofluorines by fermentation.

Combinatorial biosynthesis with other halogenases may be more challenging, Rohr says. Most flavin-dependent and all known mononuclear iron halogenases are part of large multiprotein assembly lines in which substrate is delivered on the end of a long flexible arm to each enzyme in the assembly line. It may prove difficult to use such halogenases combinatorially, he says.

Knowing how the mononuclear iron halogenases work "will undoubtedly pave the way for novel organometallic halogenation catalysts for chemical synthesis," according to chemists Nathan A. Schnarr and Chaitan Khosla of Stanford University, writing in a Nature commentary (2005, 436, 1094).

That prediction is already happening. For example, inorganic chemist Lawrence Que Jr. of the University of Minnesota previously reported that a mononuclear iron complex reminiscent of these halogenases' active site is capable of halogenating cyclohexane. Que tells C&EN that his lab is now working on model compounds that re-create these halogenases' key highly reactive oxo-iron species. Such compounds—and perhaps even the enzymes that inspired them—could someday turn out to be valuable halogenation tools for synthetic chemists.

 
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