Mechanism, inhibition and recent evolution of an unusual, promiscuous reductase
September 15-19, 2019
Dihydrofolate reductases (DHFR) catalyze the metabolically-essential reduction of dihydrofolate in an early step of purine biosynthesis. DHFRs are a central target in the control of proliferative diseases, including microbial infections. Trimethoprim is an effective antibiotic with broad clinical utility worldwide, acting as a selective inhibitor of microbial DHFRs. However, its clinical utility is threatened by the emergence of Type II microbial dihydrofolate reductases (DfrB) that are natively trimethoprim-resistant. DfrBs confer transmissible antibiotic resistance in humans and livestock. Their unusual structure and mode of substrate binding will be presented. We have created and screened active-site combinatorial libraries of a DfrB to provide key insights into the mechanism of this enzymatic reduction (1, 2). Attempts to identify promiscuous substrates for reduction have provided leads for inhibitor design. To this effect, we initiated a drug discovery program for these emerging targets. Fragment-based inhibitor development led to design of symmetrical bis-benzimidazoles that exhibit micromolar inhibition of DfrB1 (3). We determined that all closely-related Type II DHFRs share similar activity and inhibition patterns, broadening the utility of these inhibitors to the entire enzyme class. Upon whole-genome sequencing of trimethoprim-resistant E. coli from clinical isolates, we identified a dfrB gene flanked by multiple resistance genes, supporting its clinical emergence (4). To further investigate the evolutionary origin of this resistance enzyme, we undertook metagenomic screening. We have identified distantly-related structural homologs which, despite sharing only weak sequence homology, exhibit a high potential for conferring bacterial trimethoprim resistance.
Joelle Pelletier, "Mechanism, inhibition and recent evolution of an unusual, promiscuous reductase" in "Enzyme Engineering XXV", Huimin Zhao, University of Illinois at Urbana-Champaign, USA John Wong, Pfizer, USA Eds, ECI Symposium Series, (2019). https://dc.engconfintl.org/enzyme_xxv/132