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New recipes for biocatalysis

The bacterial halogenase SyrB2 catalyzes selective installation of halogens in place of unactivated aliphatic C-H bonds. By substituting halide reagents with the nitrogenous anions N3– and NO2–, SyrB2 can perform C-N bond formation reactions not previously observed in nature.

Eric M Brustad


© 2014 Nature America, Inc. All rights reserved.


nzymes are becoming increasingly important tools for chemical synthesis owing in part to their exquisite selectivity and mild reaction conditions1. Selective functionalization of unactivated C-H bonds is a hallmark example where enzymes outpace current chemical catalysis but also provide inspiration for synthetic advances2. This is underscored by numerous efforts to design biomimetic metal–ligand complexes, which in turn can provide insight into metalloenzyme function and yield synthetic alternatives to biocatalysts. Adapting reactions discovered through chemical ingenuity to biology remains challenging, and many synthetic transformations have no biological counterpart. In nature, for instance, C-N bonds are typically formed through the transamination of carbonyl substrates, whereas a compelling synthetic strategy, the direct catalytic amination of C-H bonds, is a particular example where biological variations are almost entirely absent. Matthews et al.3 now take a step toward filling this gap by demonstrating that a bacterial aliphatic halogenase SyrB2 can be coaxed to install nitrogenous anions in place of unactivated aliphatic C-H bonds, generating C-N bonds through chemistry not previously observed in nature. Inspiration for enzyme design can be taken from the paths nature follows to evolve biocatalysts. A growing consensus points to fortuitous side reactions that take place in active sites but depart from primary enzymatic functiona concept called ‘promiscuity’as central in developing new biological reactions in nature or in the lab4. Promiscuous reactions, which share common mechanistic features with native enzyme catalysis, are often very weak but can serve as evolutionary starting pointsthrough gene duplication, mutation and selectionwhen an organism is introduced into an environment where new chemistry may provide a selective advantage. The natural reagent pool, however, is limited to molecules cells can synthesize or acquire from their environment. As chemists, we have the ability to search out 170

iron-bound hydroxide accounts for ultimate substrate hydroxylation. Previous work by these authors5 provided mechanistic insight into SyrB2’s divergent ability to perform halogenation in lieu of hydroxylation: an alanine mutation results in the loss of a coordinating aspartate ligand, conserved in hydroxylases, providing space for an iron-bound halide. In addition, positioning of the carbon radical intermediate is altered in halogenases to optimize attack on the halide. On the basis of their mechanistic observations, Matthews et al.3 suspected

promiscuous reactivity that has yet to be sampled by nature due to a lack of the necessary ingredients. The Fe(ii)-dependent halogenase SyrB2 catalyzes selective installation of halides in place of unactivated C-H bonds and is homologous in both structure and mechanism to hydroxylase enzymes that catalyze C-O bond formations. Both enzyme families share a high-valent Fe(iv)-oxo intermediate capable of abstracting aliphatic hydrogen atoms, yielding a carbon-centered radical and Fe(iii)-OH adduct (Fig. 1). In hydroxylases, radical coupling to the H2N R1SOC



O Fe(IV)



Hydrogen abstraction H2N R1SOC H2N R1SOC

Hydroxylase OH

X = Asp e nas

e log – Ha Cl X=


Cl Natural biocatalysis

X His

OH Fe(III) R2 His

Halogenase X = N 3–


oge nas X= e NO – 2


N N+


N– O– N+

Non-natural biocatalysis O

Figure 1 | Homologous hydroxylase and hydrogenase enzymes share a common high valent Fe(IV)-oxo reactive intermediate capable of aliphatic hydrogen abstraction. In nature, the resulting carbon radical will recombine with an iron-bound hydroxide (hydroxylase) or chloride (halogenase) in a manner dependent on the identity of the cis-iron ligand (X, green box). Replacing the chloride ligand in halogenase SyrB2 (structure shown; Protein Data Bank code 2FCT) with the nitrogenous anions N3– or NO2– results in C-N bond coupling reactions not previously observed in nature (salmon box). R1, SyrB1 carrier protein; R2, carboxylate cosubstrate. nature chemical biology | VOL 10 | MARCH 2014 |


© 2014 Nature America, Inc. All rights reserved.

news & views that SyrB2 might catalyze promiscuous reactions if native halides were substituted with other ions. Reconstitution of the SyrB2 system, including the SyrB1 carrier protein loaded with a model unactivated aliphatic substrate, l-2-aminobutyrate, showed that SyrB2 can indeed install alternative anions in particular, N3– and NO2–  in place of a halide. Although yields using wild-type SyrB2 were very low (~4% and 8%, respectively), formation of alkylazides and nitroalkanes in this fashion is unprecedented in biology. Addition of a glycine mutation designed to create more space for the larger N3– and NO2– ions respectively increased activity to ~20% and 50% and suggests that further improvements may be achievable through evolution. Fortuitously, introduction of this glycine also reduced native halogenation activity and inhibition by contaminating chloride, a feature that may mimic respecialization toward promiscuous enzyme function in nature. Equilibrium binding experiments showed that a variety of additional anions, including CN–, OCN–, HS– and HCO2–, are also capable of accessing the halide-binding site. Although no C-H activation products were observed with these reagents, other coupling reactions may be achievable through engineering or by exploring alternative halogenase scaffolds.

Despite the novelty of this enzymatic C-N bond formation reaction, there remain severe limitations, including low product yield, that mitigate the current usefulness of this approach for chemical synthesis. For example, all of the halogenases in this family require substrates covalently attached to accessory carrier proteins (for example, SyrB1), which substantially limits substrate scope. Nevertheless, two additional SyrB1 substrates, threonine and norvaline, also show detectable levels of nitration, indicating that SyrB2 can access alternative substrates if given the chance. These challenges offer exciting opportunities for continued exploration. The power of this work highlights how detailed mechanistic insight, informed by chemical ingenuity, can provide access to new modes of biocatalysis. In this simplified approach, new chemistry may be achieved by merely interrogating natural enzymes, particularly metalloenzymes, with reagents that are rare or may not exist in the environment. This is in line with recent discoveries by Arnold and associates6–8 showing that cytochrome P450s can access metallocarbenoid and nitrenoid insertion reactions when provided with non-natural diazoacetate and sulfonazide reagents. Alternative strategies for engineering new enzyme activity through the immobilization of non-natural

metallocenters have gained traction in recent years but have been limited to highly specialized protein scaffolds9,10. Approaches that access new reactivity through the promiscuous use of synthetic reagents may markedly expand our ability to engineer non-natural catalysis by making use of the vast diversity of enzymes that already exist in nature. ■ Eric M. Brustad is at the Department of Chemistry, Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. e-mail: [email protected] Published online 26 January 2014 doi:10.1038/nchembio.1457 References

1. Bornscheuer, U.T. et al. Nature 485, 185–194 (2012). 2. Chen, M.S. & White, M.C. Science 318, 783–787 (2007). 3. Matthews, M.L. et al. Nat. Chem. Biol. doi:10.1038/nchembio.1438 (26 January 2014). 4. Khersonsky, O. & Tawfik, D.S. Annu. Rev. Biochem. 79, 471–505 (2010). 5. Matthews, M.L. et al. Proc. Natl. Acad. Sci. USA 106, 17723–17728 (2009). 6. Coelho, P.S. et al. Science 339, 307–310 (2013). 7. McIntosh, J.A. et al. Angew. Chem. Int. Ed. Engl. 52, 9309–9312 (2013). 8. Wang, Z.J. et al. Chem. Sci. 5, 598–601 (2014). 9. Wilson, M.E. & Whitesides, G.M. J. Am. Chem. Soc. 100, 306–307 (1978). 10. Hyster, T.K. et al. Science 338, 500–503 (2012).

Competing financial interests The author declares no competing financial interests.


Pain enters through the side door

TRPM3 can permeate ions through two distinct pores—the central pore and a likely alternative ‘omega pore’. Entry of ions through the alternative pore of TRPM3 contributes to pain generation, making it an attractive target for the design of new analgesics.

Emily R Liman


he prevailing dogma and an abundance of evidence tells us that voltage-gated ion channels contain a single central permeation pathway formed at the juncture of the four channel subunits (or pseudosubunits). This conclusion, supported by crystallographic data from channels as divergent as voltage-gated K+ channels1 and ligand- and heat-gated transient receptor potential (TRP) channels2, has important implications for the development of pharmacological agents that block ion entry. Now, Vriens et al.3 challenge this ‘central pore dogma’ by showing that the ion channel TRPM3 can conduct ions through an

alternative pathway opened upon exposure to specific chemical signals. TRPM3 is a member of the family of TRP ion channels, a family that contains a number of ion channels involved in sensory signaling. TRPM3, which was first cloned in 2003, remained an orphan channel, without a ligand or validated function4, until it was discovered that the neuroactive steroid pregnelone sulfate (PS) was a potent activator of the channel5. Although the conditions under which elevated PS levels gate the channel are not known, the availability of a validated agonist has allowed detailed study of

nature chemical biology | VOL 10 | MARCH 2014 |

TRPM3. Most surprisingly, TRPM3 was found to be expressed in nociceptors, where, along with the better-studied TRP channel family members TRPV1 and TRPA1, it contributes to the generation of pain6. Accordingly, intraplantar injection of PS elicits nocifensive behavior not observed in TRPM3 knockout mice. Indeed, hypersensitivity in response to inflammatory mediators is reduced in TRPM3 knockout animals, to a similar extent as observed in TRPV1 knockout animals. But here is where the story gets a little bit strange. In search of TRPM3 171

C-H activation: New recipes for biocatalysis.

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