Characterization of the Three DHFRs and K65P Variant: Enhanced Substrate Affinity and Molecular Dynamics Analysis

Dihydrofolate reductase (DHFR) is ubiquitously present in all living organisms and plays a crucial role in the growth of the fungal pathogen R.solani. Sequence alignment confirmed the evolutionary conservation of the essential lid domain, with the amino acid ‘P’ within the PEKN lid domain appearing with a frequency of 89.5% in higher organisms and 11.8% in lower organisms. Consequently, a K65P variant was introduced into R.solani DHFR (rDHFR). Subsequent enzymatic kinetics assays were conducted for human DHFR (hDHFR), rDHFR, E. coli DHFR (eDHFR), and the K65P variant. hDHFR exhibited the highest k_cat of 0.95 s⁻¹, followed by rDHFR with 0.14 s⁻¹, while eDHFR displayed the lowest k_cat of 0.09 s⁻¹. Remarkably, the K65P variant induced a significant reduction in K_m, resulting in a 1.8-fold enhancement in catalytic efficiency (k_cat/K_m) relative to the wild type. Differential scanning fluorimetry and binding free energy calculations confirmed the enhanced substrate affinity for both folate and NADPH in the K65P variant. These results suggest that the K65P mutation enhances substrate affinity and catalytic efficiency in DHFR, highlighting the evolutionary and functional importance of the K65 residue.