Structure-based switch of regioselectivity in the flavin-dependent tryptophan 6-halogenase Thal
| Autor: | Ann-Christin Moritzer, Norbert Sewald, Hannah Minges, Tina Prior, Marcel Frese, Hartmut H. Niemann |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Stereochemistry Protein design Flavoprotein Flavin group Crystallography X-Ray Biochemistry Substrate Specificity 03 medical and health sciences Structure-Activity Relationship Bacterial Proteins Catalytic Domain ddc:610 Binding site Molecular Biology tryptophan halogenase Indole test 030102 biochemistry & molecular biology biology Chemistry Tryptophan Active site Cell Biology Enzyme structure Streptomyces 030104 developmental biology biology.protein Enzymology Oxidoreductases |
| Zdroj: | JBC papers in press 294(7), 2529-2542 (2018). doi:10.1074/jbc.RA118.005393 |
| DOI: | 10.1074/jbc.RA118.005393 |
| Popis: | JBC papers in press 294(7), 2529 - 2542 (2019). doi:10.1074/jbc.RA118.005393 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. Published by American Soc. for Biochemistry and Molecular Biology, Bethesda, MD. |
| Databáze: | OpenAIRE |
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