Recent Advances in Synthetic Application and Engineering of Halogenases

Minges H, Sewald N. Recent Advances in Synthetic Application and Engineering of Halogenases. ChemCatChem. 2020;12(18):4450-4470.Halogenating enzymes are able to introduce halogen substituents under ambient conditions using non-hazardous reagents with intriguing selectivity, which is highly desired in green chemistry. Although C-H functionalization such as halogenation is a well-known transformation in synthetic chemistry, the selective incorporation of halogens using conventional chemical approaches often remains challenging. Therefore, enzyme-based strategies have been emerging as valuable alternatives in recent years. Inspired by manifold developments of enzymatic halogenation, this review focuses on advances of halogenating enzymes and their application with particular emphasis on FAD-dependent halogenases (FDHs). Catalytic strategies, application scope and engineering of FDHs are outlined pointing to the increasing utility of halogenases as promising biocatalysts. Current limitations as well as potential future developments of their synthetic utility are being discussed

Anti-Markovnikov alkene oxidation by metal-oxo–mediated enzyme catalysis

Catalytic anti-Markovnikov oxidation of alkene feedstocks could simplify synthetic routes to many important molecules and solve a long-standing challenge in chemistry. Here we report the engineering of a cytochrome P450 enzyme by directed evolution to catalyze metal-oxo–mediated anti-Markovnikov oxidation of styrenes with high efficiency. The enzyme uses dioxygen as the terminal oxidant and achieves selectivity for anti-Markovnikov oxidation over the kinetically favored alkene epoxidation by trapping high-energy intermediates and catalyzing an oxo transfer, including an enantioselective 1,2-hydride migration. The anti-Markovnikov oxygenase can be combined with other catalysts in synthetic metabolic pathways to access a variety of challenging anti-Markovnikov functionalization reactions

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Engineering of Tryptophan Halogenases

Minges H. Engineering of Tryptophan Halogenases. Bielefeld: Universität Bielefeld; 2020

A High-Throughput Fluorescence Assay to Determine the Activity of Tryptophan Halogenases

Schnepel C, Minges H, Frese M, Sewald N. A High-Throughput Fluorescence Assay to Determine the Activity of Tryptophan Halogenases. Angewandte Chemie International Edition. 2016;55(45):14159-14163.Biocatalytic halogenation with tryptophan halogenases is hampered by severe limitations such as low activity and stability. These drawbacks can be overcome by directed evolution, but for screening large mutant libraries, a facile high-throughput method is required. Therefore, we developed a quantitative halogenase assay based on a Suzuki-Miyaura cross-coupling towards the formation of a fluorescent aryltryptophan. The technique was optimized for application in crude E.coli lysate without intermediary purification steps, and was used for quantitatively monitoring the formation of halogenated tryptophans with high specificity by facile fluorescence screening in microtiter plates. This novel screening approach was exploited to engineer a thermostable tryptophan 6-halogenase. Libraries were constructed by error-prone PCR and selected for improved thermal resistance simply by fluorogenic cross-coupling. Our method led to an enzyme variant with substantially increased thermal stability and 2.5-fold improved activity

Ein Hochdurchsatz-Fluoreszenz-Assay zur Bestimmung der Aktivität von Tryptophan-Halogenasen

Schnepel C, Minges H, Frese M, Sewald N. Ein Hochdurchsatz-Fluoreszenz-Assay zur Bestimmung der Aktivität von Tryptophan-Halogenasen. Angewandte Chemie. 2016;128(45):14365-14369

Metabolic engineering of Corynebacterium glutamicum for the fermentative production of halogenated tryptophan

Veldmann K, Minges H, Sewald N, Lee J-H, Wendisch VF. Metabolic engineering of Corynebacterium glutamicum for the fermentative production of halogenated tryptophan. Journal of Biotechnology. 2019;291:7-16

Structure-based switch of regioselectivity in the flavin-dependent tryptophan 6-halogenase Thal

Moritzer A-C, Minges H, Prior T, Frese M, Sewald N, Niemann H. Structure-based switch of regioselectivity in the flavin-dependent tryptophan 6-halogenase Thal. Journal of Biological Chemistry. 2019;294(7):2529-2542.Flavin-dependent halogenases increasingly attract attention as biocatalysts in organic synthesis, facilitating environmentally friendly halogenation strategies that require only FADH(2), oxygen, and halide salts. Different flavin-dependent tryptophan halogenases regioselectively chlorinate or brominate trypto-phan's indole moiety at C5, C6, or C7. Here, we present the first substrate-bound structure of a tryptophan 6-halogenase, namely Thal, also known as ThdH, from the bacterium Streptomyces albogriseolus at 2.55 angstrom resolution. The structure revealed that the C6 of tryptophan is positioned next to the epsilon-amino group of a conserved lysine, confirming the hypothesis that proximity to the catalytic residue determines the site of electrophilic aromatic substitution. Although Thal is more similar in sequence and structure to the tryptophan 7-halogenase RebH than to the tryptophan 5-halogenase PyrH, the indole binding pose in the Thal active site more closely resembled that of PyrH than that of RebH. The difference in indole orientation between Thal and RebH appeared to be largely governed by residues positioning the Trp backbone atoms. The sequences of Thal and RebH lining the substrate binding site differ in only few residues. Therefore, we exchanged five amino acids in the Thal active site with the corresponding counterparts in RebH, generating the quintuple variant Thal-RebH5. Overall conversion of l-Trp by the Thal-RebH5 variant resembled that of WT Thal, but its regioselectivity of chlorination and bromination was almost completely switched from C6 to C7 as in RebH. We conclude that structure-based protein engineering with targeted substitution of a few residues is an efficient approach to tailoring flavin-dependent halogenases

Structure-based switch of regioselectivity in the flavin-dependent tryptophan 6-halogenase Thal.

Flavin-dependent halogenases increasingly attract attention as biocatalysts in organic synthesis, facilitating environmentally friendly halogenation strategies that require only FADH2, oxygen, and halide salts. Different flavin-dependent tryptophan halogenases regioselectively chlorinate or brominate trypto-phan's indole moiety at C5, C6, or C7. Here, we present the first substrate-bound structure of a tryptophan 6-halogenase, namely Thal, also known as ThdH, from the bacterium Streptomyces albogriseolus at 2.55 Å resolution. The structure revealed that the C6 of tryptophan is positioned next to the ϵ-amino group of a conserved lysine, confirming the hypothesis that proximity to the catalytic residue determines the site of electrophilic aromatic substitution. Although Thal is more similar in sequence and structure to the tryptophan 7-halogenase RebH than to the tryptophan 5-halogenase PyrH, the indole binding pose in the Thal active site more closely resembled that of PyrH than that of RebH. The difference in indole orientation between Thal and RebH appeared to be largely governed by residues positioning the Trp backbone atoms. The sequences of Thal and RebH lining the substrate binding site differ in only few residues. Therefore, we exchanged five amino acids in the Thal active site with the corresponding counterparts in RebH, generating the quintuple variant Thal-RebH5. Overall conversion of l-Trp by the Thal-RebH5 variant resembled that of WT Thal, but its regioselectivity of chlorination and bromination was almost completely switched from C6 to C7 as in RebH. We conclude that structure-based protein engineering with targeted substitution of a few residues is an efficient approach to tailoring flavin-dependent halogenases

Targeted Enzyme Engineering Unveiled Unexpected Patterns of Halogenase Stabilization

Minges H, Schnepel C, Boettcher D, et al. Targeted Enzyme Engineering Unveiled Unexpected Patterns of Halogenase Stabilization. ChemCatChem. 2020;12(3):818-831.Halogenases are valuable biocatalysts for selective C-H activation, but despite recent efforts to broaden their application scope by means of protein engineering, improvement of thermostability and catalytic efficiency is still desired. A directed evolution campaign aimed at generating a thermostable flavin-dependent tryptophan 6-halogenase with reasonable activity suitable for chemoenzymatic purposes. These characteristics were tackled by combining successive rounds of epPCR along with semi-rational mutagenesis leading to a triple mutant (Thal-GLV) with substantially increased thermostability (T-M=23.5 K) and higher activity at 25 degrees C than the wild type enzyme. Moreover, an active-site mutation has a striking impact on thermostability but also on enantioselectivity. Our data contribute to a detailed understanding of biohalogenation and provide a profound basis for future engineering strategies to facilitate chemoenzymatic application of these attractive biocatalysts