Mutational analysis confirms the presence of distal inhibitor-selectivity determining residues in B. stearothermophilus dihydrofolate reductase

• Published in: Archives of biochemistry and biophysics 2020-10, Vol.692, p.108545-108545, Article 108545
• Main Authors: Eck, Tyler, Patel, Seema, Candela, Thomas, Leon H, Katherine, Little, Michael, Reis, Natalia E., Liyanagunawardana, Uththara, Gubler, Ueli, Janson, Cheryl A., Catalano, Jaclyn, Goodey, Nina M.
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Bacterial Proteins - antagonists & inhibitors; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; DHFR; Distal residues; Drug binding; Folic Acid Antagonists - chemistry; Geobacillus stearothermophilus - enzymology; Geobacillus stearothermophilus - genetics; Inhibitor selectivity; Mutation, Missense; Pyrimethamine; Tetrahydrofolate Dehydrogenase - chemistry; Tetrahydrofolate Dehydrogenase - genetics; Trimethoprim
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2020.108545

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. [Display omitted] •Asp27 is a catalytically critical active site residue in B. stearothermophilus DHFR.•The residue cluster His38, Ala39, Ile40 is a predicted DHFR selectivity determinant.•Mutations in the cluster significantly impacted inhibitor binding and selectivity.•Most cluster mutations increased selectivity for pyrimethamine over trimethoprim.