Enhanced Solubilization of Class B Radical S-Adenosylmethionine Methylases by Improved Cobalamin Uptake in Escherichia coli
| Autor: | Erin L. McCarthy, Anthony J. Blaszczyk, Nicholas D. Lanz, Squire J. Booker, Bo Wang, Brianne S. Jones, Roy X. Wang |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
S-Adenosylmethionine 010402 general chemistry medicine.disease_cause 01 natural sciences Biochemistry Cobalamin Cofactor Article 03 medical and health sciences chemistry.chemical_compound Biosynthesis medicine Escherichia coli Overproduction chemistry.chemical_classification biology Methylation Methyltransferases 0104 chemical sciences Vitamin B 12 030104 developmental biology Enzyme chemistry Solubility biology.protein Radical SAM |
| Zdroj: | Biochemistry. 57(9) |
| ISSN: | 1520-4995 |
| Popis: | The methylation of unactivated carbon and phosphorus centers is a burgeoning area of biological chemistry, especially given that such reactions constitute key steps in the biosynthesis of numerous enzyme cofactors, antibiotics, and other natural products of clinical value. These kinetically challenging reactions are catalyzed exclusively by enzymes in the radical S-adenosylmethionine (SAM) superfamily and have been grouped into four classes: A, B, C, and D. Class B radical SAM (RS) methylases require a cobalamin cofactor in addition to the [4Fe-4S] cluster that is characteristic of RS enzymes. However, their poor solubility upon overexpression and their generally poor turnover has hampered detailed in vitro studies of these enzymes. It has been suggested that improper folding, possibly caused by insufficient cobalamin during their overproduction in Escherichia coli, leads to formation of inclusion bodies. Herein, we report our efforts to improve the overproduction of class B RS methylases in a soluble form by engineering a strain of E. coli to take in more cobalamin. We cloned five genes (btuC, btuE, btuD, btuF, and btuB) that encode proteins that are responsible for cobalamin uptake and transport in E. coli and coexpressed these genes with those that encode TsrM, Fom3, PhpK, and ThnK, four class B RS methylases that suffer from poor solubility during overproduction. This strategy markedly enhances cobalamin uptake into the cytoplasm and improves the solubility of the target enzymes significantly. |
| Databáze: | OpenAIRE |
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