Mutational analysis confirms the presence of distal inhibitor-selectivity determining residues in B. stearothermophilus dihydrofolate reductase
| Autor: | Ueli Gubler, Uththara Liyanagunawardana, Thomas Candela, Nina M. Goodey, Jaclyn Catalano, Natalia E. Reis, Michael Little, Katherine Leon H, Tyler Eck, Seema Patel, Cheryl A. Janson |
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| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Stereochemistry Mutation Missense Biophysics Biochemistry Geobacillus stearothermophilus 03 medical and health sciences Residue (chemistry) Bacterial Proteins Dihydrofolate reductase Enzyme kinetics Molecular Biology chemistry.chemical_classification 030102 biochemistry & molecular biology biology Active site Ligand (biochemistry) Amino acid Tetrahydrofolate Dehydrogenase 030104 developmental biology Enzyme Amino Acid Substitution chemistry biology.protein Folic Acid Antagonists Selectivity |
| Zdroj: | Archives of Biochemistry and Biophysics. 692:108545 |
| ISSN: | 0003-9861 |
| Popis: | Many antibacterial and antiparasitic drugs work by competitively inhibiting dihydrofolate reductase (DHFR), a vital enzyme in folate metabolism. The interactions between inhibitors and DHFR active site residues are known in many homologs but the contributions from distal residues are less understood. Identifying distal residues that aid in inhibitor binding can improve targeted drug development programs by accounting for distant influences that may be less conserved and subject to frequent resistance causing mutations. Previously, a novel, homology-based, computational approach that mines ligand inhibition data was used to predict residues involved in inhibitor selectivity in the DHFR family. Expectedly, some inhibitor selectivity determining residue positions were predicted to lie in the active site and coincide with experimentally known inhibitor selectivity determining positions. However, other residues that group spatially in clusters distal to the active site have not been previously investigated. In this study, the effect of introducing amino acid substitutions at one of these predicted clusters (His38-Ala39-Ile40) on the inhibitor selectivity profile in Bacillus stearothermophilus dihydrofolate reductase (Bs DHFR) was investigated. Mutations were introduced into these cluster positions to change sidechain chemistry and size. We determined kcat and KM values and measured KD values at equilibrium for two competitive DHFR inhibitors, trimethoprim (TMP) and pyrimethamine (PYR). Mutations in the His38-Ala39-Ile40 cluster significantly impacted inhibitor binding and TMP/PYR selectivity - seven out of nine mutations resulted in tighter binding to PYR when compared to TMP. These data suggest that the His38-Ala39-Ile40 cluster is a distal inhibitor selectivity determining region that favors PYR binding in Bs DHFR and, possibly, throughout the DHFR family. |
| Databáze: | OpenAIRE |
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