Halogenation and Dehalogenation

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

doi:10.1002/9781118697856.ch07

Cited by 3 publications

(5 citation statements)

References 34 publications

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“…Related enzymes that chlorinate the 6- and 5-positions of tryptophan have also been characterized, however (Thal and PyrH, respectively), 36 suggesting that it should be possible to alter RebH selectivity. Furthermore, site-directed mutagenesis of the 7-halogenase PrnA led to a variant that provided a 1 : 2 mixture of 5- and 7-chlorotryptophans, 37 and a similar approach was used to alter the selectivity of PrnA toward 2-aminobenzoic acid so that 5-chlorination was favored over 3-chlorination (from 84 : 16 for PrnA to 38 : 62). 38 While these examples show that halogenase selectivity can be altered, low selectivities were observed, and an initial examination of PrnA substrate scope 39 indicated that substituted indoles were chlorinated on the pyrrole ring.…”

Section: Resultsmentioning

confidence: 99%

“… 62 The same subtleties that complicate such analysis lead to the difficulty of rationally introducing specific mutations to improve enzyme function. 24 Indeed, earlier attempts to modify the selectivity of PrnA toward tryptophan 37 or substituted benzenes 38 by mutating active site residues led to enzymes with poor selectivity, and active site mutations introduced into RebH to alter its specificity from tryptophan to tryptamine did not change its selectivity. 50 …”

Section: Resultsmentioning

confidence: 99%

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Directed evolution of RebH for catalyst-controlled halogenation of indole C–H bonds

et al. 2016

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RebH variants capable of chlorinating substituted indoles ortho-, meta-, and para- to the indole nitrogen were evolved by directly screening for altered selectivity on deuterium-substituted probe substrates using mass spectrometry. This systematic approach allowed for rapid accumulation of beneficial mutations using simple adaptive walks and should prove generally useful for altering and optimizing the selectivity of C-H functionalization catalysts. Analysis of the beneficial mutations showed that structure-guided selection of active site residues for targeted mutagenesis can be complicated either by activity/selectivity tradeoffs that reduce the possibility of detecting such mutations or by epistatic effects that actually eliminate the benefits of a mutation in certain contexts. As a corollary to this finding, the precise manner in which the beneficial mutations identified led to the observed changes in RebH selectivity is not clear. Docking simulations suggest that tryptamine binds to these variants as tryptophan does to native halogenases, but structural studies will be required to confirm these models and shed light on how particular mutations impact tryptamine binding. Similar directed evolution efforts on other enzymes or artificial metalloenzymes could enable a wide range of C-H functionalization reactions.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Directed evolution of RebH for catalyst-controlled halogenation of indole C–H bonds

et al. 2016

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“…While the FAD binding site is well conserved among different FDHs, a significant difference lies in the substrate binding site which allows FDHs to utilize a broad range of substrates and catalyze halogenation at various positions (14). Therefore, most of the previous studies have been focused on enzyme engineering to alter substrate scope and regio-specificity (14,(20)(21)(22)(23)(24)(25)(26), while less attention has been paid to mechanistic investigation.…”

Section: Introductionmentioning

confidence: 99%

Dissecting the low catalytic capability of flavin-dependent halogenases

Phintha

Prakinee

Jaruwat

et al. 2021

Journal of Biological Chemistry

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Although flavin-dependent halogenases (FDHs) are attractive biocatalysts, their practical applications are limited because of their low catalytic efficiency. Here, we investigated the reaction mechanisms and structures of tryptophan 6-halogenase (Thal) from Streptomyces albogriseolususing stopped-flow, rapid-quench flow, QM/MM calculations, crystallography and detection of intermediate (hypohalous acid (HOX)) liberation. We found that the key flavin intermediate, C4a-hydroperoxyflavin (C4aOOH-FAD), formed by Thal and other FDHs(tryptophan 7-halogenase (PrnA) and tryptophan 5-halogenase (PyrH)), can react with I-, Br-, and Cl-but not F-, to form C4a-hydroxyflavin and HOX. Our experiments revealed that I-reacts with C4aOOH-FAD the fastest with the lowest energy barrier, and have shown for the first time that a significant amount of the HOX formed leaks out as free HOX. This leakage is probably a major cause of low product coupling ratios in all FDHs. Site-saturation mutagenesis of Lys79 showed that changing Lys79 to any other amino acid resulted in an inactive enzyme. However, the levels of liberated HOX of these variants are all similar, implying that Lys79 probably does not form a chloramine or bromamine intermediateas previously proposed. Computational calculations revealed that Lys79 has an abnormally lower pKa compared to other Lys residues, implying that the catalytic Lys may act as a proton donor in catalysis. Analysis of new X-ray structures of Thal also explains why pre-mixing of FDHs with FADH- generally results in abolishment of C4aOOH-FAD formation. These findings reveal the hidden factors restricting FDHs capability which should be useful for future development of FDHs applications.

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“…The kinetic parameters were determined for PrnC with K M = 14.40 ± 1.20 µM and k cat = 1.66 ± 0.02 min -1 (k cat /K M = 0.115 ± 0.011 μM -1 min -1 ) due to synthesis of the natural product (4) and calibration (see Supplementary Section S1.5.5). These parameters were in range with those of PrnA (Lang et al, 2011;Minges and Sewald, 2020a), RebH (Dachwitz et al, 2020), and RadH (Menon et al, 2017), ranging from 0.0006 μM -1 min -1 to 12 μM -1 min -1 for various substrates, but pyrroles have not been reported. As the synthesis route of APRN (4) has a few bottlenecks and needs relatively high effort, we also wanted to compare the chemical late-stage halogenation reactions to the biocatalysis.…”

Section: Discussionmentioning

confidence: 71%

“…Fl-Hals have already entered research stages of applicability rather than sheer understanding. Site-directed mutagenesis could vary regioselectivity (Lang et al, 2011;Shepherd et al, 2015;Andorfer and Lewis, 2018), and even upscaling by the use of cross-linked enzyme aggregates (CLEAs) to gram-scale conversions were possible (Frese and Sewald, 2015b;a). Finally, follow-up chemistry has already been carried out such as crosscoupling reactions in crude lysate (Gruß and Sewald, 2020).…”

Section: Introductionmentioning

confidence: 99%

Expression and characterization of PrnC—a flavin-dependent halogenase from the pyrrolnitrin biosynthetic pathway of Pseudomonas protegens Pf-5

2023

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Introduction: The antimicrobial pyrrolnitrin from Pseudomonas strains is formed in four steps from tryptophan and comprises two flavin-dependent halogenases. Both PrnC and PrnA can carry out regioselective chlorination and bromination and are carrier protein-independent. Whilst the tryptophan halogenase PrnA has been studied in detail in the past, this study focuses on the pyrrole halogenating enzyme PrnC.Methods: The halogenating enzyme PrnC, as well as the essential electron suppliers, the flavin reductases, have been produced soluble in E. coli. Furthermore, a screening of a rational compound library revealed that the pyrrole is essential for substrate recognition; however, the substitution pattern of the benzene ring is not limiting the catalysis.Results and discussion: This renders PrnC to be a synthetically valuable enzyme for the synthesis of pyrrolnitrin congeners. For its natural substrate monodechloroaminopyrrolnitrin (MDA), the KM value was determined as 14.4 ± 1.2 µM and a kcat of 1.66 ± 0.02 min−1, which is comparable to other halogenases.

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Halogenation and Dehalogenation

Cited by 3 publications

References 34 publications

Directed evolution of RebH for catalyst-controlled halogenation of indole C–H bonds

Directed evolution of RebH for catalyst-controlled halogenation of indole C–H bonds

Dissecting the low catalytic capability of flavin-dependent halogenases

Expression and characterization of PrnC—a flavin-dependent halogenase from the pyrrolnitrin biosynthetic pathway of Pseudomonas protegens Pf-5

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