Expression, purification, and characterization of transmembrane protein homogentisate solanesyltransferase

Homogentisate solanesyltransferase (HST) is a crucial enzyme in the plastoquinone biosynthetic pathway and has recently emerged as a promising target for herbicides. In this study, we successfully expressed and purified a stable and highly pure form of seven times transmembrane protein Chlamydomonas reinhardtii HST ( Cr HST). The final yield of Cr HST protein obtained was 12.2 mg per liter of M9 medium. We evaluated the inhibitory effect on Cr HST using Des-Morpholinocarbony Cyclopyrimorate (DMC) and found its IC 50 value to be 3.63 ± 0.53 μM, indicating significant inhibitory potential. Additionally, we investigated the substrate affinity of Cr HST with two substrates, determining the K m values as 22.76 ± 1.70 μM for FPP and 48.54 ± 3.89 μM for HGA. Through sequence alignment analyses and three-dimensional structure predictions, we identified conserved amino acid residues forming the active cavity in the enzyme. The results from molecular docking and binding energy calculations indicate that DMC has a greater binding affinity with HST compared to HGA. These findings represent substantial progress in understanding Cr HST’s properties and potential for herbicide development. Key points • First high-yield transmembrane CrHST protein via E. coli system • Preliminarily identified active cavity composition via activity testing • Determined substrate and inhibitor modes via molecular docking