Preliminary study of the lethal fungus Candida albicans phosphofructokinase-1 as a potential therapeutic target.

Our previous research revealed that phosphofructokinase-1 (Pfk1) is a key regulator in fungal virulence [1]. However, the unclear structural characterization and catalytic mechanisms hinder the research on whether it can become a potential antifungal drug target. Here, we report the first purification of Pfk1 from the lethal fungal C. albicans using affinity and ion exchange chromatography, yielding two 100 kDa protein bands. Mass spectrometry identified these bands as the α- and β-subunits. Interestingly, the α subunit has serine at position 164, which differs from the asparagine shown in the previous published study. Coupled enzyme activity assays revealed that the enzyme activity of Pfk1 increased significantly from 3.09 U/mg to 38.32 U/mg during the purification process. We observed the assembly of Pfk1 via native-PAGE and found that its native active structure primarily exists as a heterooctamer with a molecular mass of 852 kDa. Further, we obtained the three-dimensional structure of C. albicans Pfk1 through homology modeling composed of four α-helices and four β-sheets arranged in a centrosymmetric manner. We identified the enzyme's binding pocket and found its amino acid sequence has low similarity to that of humans through cross-species alignment. Therefore, we performed a structure-based virtual screening of small-molecule compounds, which led to the identification of compounds that potentially target Pfk1. These compounds inhibited Pfk1 activity. Our findings preliminarily reveal the structure of lethal fungal Pfk1 and suggest that it exhibits the functional and structural characteristics of a potential therapeutic target.