Changing the Regioselectivity of the Tryptophan 7‐Halogenase PrnA by Site‐Directed Mutagenesis

Lang, Alexander; Polnick, Stefan; Nicke, Tristan; William, P; Patallo, Eugenio P.; Naismith, James H.; Pée, Karl‐Heinz van

doi:10.1002/anie.201007896

Cited by 84 publications

(76 citation statements)

References 20 publications

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“…Notably, the active site of Bmp2-TM exhibited no perturbation of the bound FAD cofactor with respect to WT Bmp2, nor did it lead to any apparent change in halogen binding properties of the enzyme, as demonstrated by conservation of its specificity for bromide. Already, for flavin-dependent tryptophan halogenases, it has been demonstrated that amino acid side chains that constitute the substrate binding site control the regiochemical outcomes for halogen additions (17,24,25 this observation to flavin-dependent halogenases that catalyze halogenation of aromatic substrates acylated to ACPs in demonstrating that side chains of residues lining the putative halogenase active site play a role in controlling substrate access, and potentially in specifying the positions on the pyrrole ring that are accessible to halogenation. Unfortunately, in the case of Mpy16, efforts to alter the putative substrate-binding cavity to resemble that of Bmp2 resulted in insolubility of the mutant Mpy16 enzymes.…”

Section: Discussionmentioning

confidence: 99%

Biosynthesis of coral settlement cue tetrabromopyrrole in marine bacteria by a uniquely adapted brominase–thioesterase enzyme pair

Gamal

Agarwal

Diethelm

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

118

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Halogenated pyrroles (halopyrroles) are common chemical moieties found in bioactive bacterial natural products. The halopyrrole moieties of mono-and dihalopyrrole-containing compounds arise from a conserved mechanism in which a proline-derived pyrrolyl group bound to a carrier protein is first halogenated and then elaborated by peptidic or polyketide extensions. This paradigm is broken during the marine pseudoalteromonad bacterial biosynthesis of the coral larval settlement cue tetrabromopyrrole (1), which arises from the substitution of the proline-derived carboxylate by a bromine atom. To understand the molecular basis for decarboxylative bromination in the biosynthesis of 1, we sequenced two Pseudoalteromonas genomes and identified a conserved four-gene locus encoding the enzymes involved in its complete biosynthesis. Through total in vitro reconstitution of the biosynthesis of 1 using purified enzymes and biochemical interrogation of individual biochemical steps, we show that all four bromine atoms in 1 are installed by the action of a single flavin-dependent halogenase: Bmp2. Tetrabromination of the pyrrole induces a thioesterase-mediated offloading reaction from the carrier protein and activates the biosynthetic intermediate for decarboxylation. Insights into the tetrabrominating activity of Bmp2 were obtained from the high-resolution crystal structure of the halogenase contrasted against structurally homologous halogenase Mpy16 that forms only a dihalogenated pyrrole in marinopyrrole biosynthesis. Structure-guided mutagenesis of the proposed substrate-binding pocket of Bmp2 led to a reduction in the degree of halogenation catalyzed. Our study provides a biogenetic basis for the biosynthesis of 1 and sets a firm foundation for querying the biosynthetic potential for the production of 1 in marine (meta)genomes.natural products | biosynthesis | halogenation | enzymology

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Section: Discussionmentioning

confidence: 99%

Biosynthesis of coral settlement cue tetrabromopyrrole in marine bacteria by a uniquely adapted brominase–thioesterase enzyme pair

Gamal

Agarwal

Diethelm

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

118

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“…As the monooxygenase screening methods reported so far are dependent upon detection of a specific reaction product or sequence of reactions occurring, they are not appropriate for application to the flavin-dependent halogenases, although methods which could be applied to their directed evolution are slowly emerging and have been demonstrated feasible. 223,240,241 In combination with what is known about the modification of their substrate scope, regioselectivity and stability through mutagenesis, 150,155,[222][223][224][225] in addition to the discovery of naturally-occurring thermostable Fl-Hals, 145 a number of reliable starting points for further engineering efforts are known.…”

Section: 113mentioning

confidence: 99%

Development of Halogenase Enzymes for Use in Synthesis

Latham

Brandenburger

Shepherd³

et al. 2017

Chem. Rev.

285

244

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Nature has evolved halogenase enzymes to regioselectively halogenate a diverse range of biosynthetic precursors, with the halogens introduced often having a profound effect on the biological activity of the resulting natural products. Synthetic endeavours to create non-natural bioactive small molecules for pharmaceutical and agrochemical applications have also arrived at a similar conclusion -halogens can dramatically improve the properties of organic molecules for selective modulation of biological targets in vivo. Consequently a high proportion of pharmaceuticals and agrochemicals on the market today possess halogens. Halogenated organic compounds are also common intermediates in synthesis and are particularly valuable in metal catalyzed cross-coupling reactions.Despite the potential utility of organohalogens, traditional non-enzymatic halogenation chemistry utilizes deleterious reagents and often lacks regiocontrol. Reliable, facile and cleaner methods for the regioselective halogenation of organic compounds are therefore essential in the development of economical and environmentally-friendly industrial processes. A potential avenue towards such methods is the use of halogenase enzymes, responsible for the biosynthesis of halogenated natural products, as biocatalysts. This review will discuss the advances towards this goal thus far, in addition to untapped potential sources of such biocatalysts and how further development of the enzymes may be focused in order to achieve the goal of industrial scale biohalogenation.

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“…Protein engineering of an FDH by rational site-specific mutagenesis was first reported in 2011 in which mutant F103A of the halogenase PrnA was found to change the strict 7-selectivity of WT to a 2 : 1 mixture of the 7-and 5-chlorinated products. 53 In other early work, rational design on the basis of structure-guided mutagenesis was applied to other FDHs with notable changes in site-selectivity in reactions of structurally different substrates, although a 100% shift in selectivity remains to be achieved. 54,55 Directed evolution of halogenases offers a more general and reliable solution to the problem of controlling the regioselectivity of halogenation.…”

Section: Engineering Site-selectivity Of Halogenasesmentioning

confidence: 99%

Enzymatic site-selectivity enabled by structure-guided directed evolution

Wang

Reetz

2017

Chem. Commun.

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Biocatalytic site-selective (regioselective) organic transformations have been practiced for decades, but the traditional limitations of enzymes regarding narrow substrate acceptance and the often observed insufficient degree of selectivity have persisted until recently. With the advent of directed evolution, it is possible to engineer site-selectivity to suit the needs of organic chemists. This review features recent progress in this exciting research area, selected examples involving P450 monooxygenases, halogenases and Baeyer-Villiger monooxygenases being featured for illustrative purposes. The complementary nature of enzymes and man-made catalysts is emphasized.

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Changing the Regioselectivity of the Tryptophan 7‐Halogenase PrnA by Site‐Directed Mutagenesis

Cited by 84 publications

References 20 publications

Biosynthesis of coral settlement cue tetrabromopyrrole in marine bacteria by a uniquely adapted brominase–thioesterase enzyme pair

Biosynthesis of coral settlement cue tetrabromopyrrole in marine bacteria by a uniquely adapted brominase–thioesterase enzyme pair

Development of Halogenase Enzymes for Use in Synthesis

Enzymatic site-selectivity enabled by structure-guided directed evolution

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