Expression, purification, and characterization of transmembrane protein homogentisate solanesyltransferase

Abstract

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 (CrHST). The final yield of CrHST protein obtained was 12.2 mg per liter of M9 medium. We evaluated the inhibitory effect on CrHST using Des-Morpholinocarbony Cyclopyrimorate (DMC) and found its IC₅₀ value to be 3.63 ± 0.53 μM, indicating significant inhibitory potential. Additionally, we investigated the substrate affinity of CrHST 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 CrHST'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.

Keywords: Bacterial expression system; Enzyme activity; Homogentisate solanesyltransferase; Membrane protein; Molecular docking.

Fig. 1

The experimental procedure for HST expression, purification, and characterization (created with MedPeer (www.medpeer.cn))

Fig. 2

Construction of CrHST into vectors. a Construction of CrHST into pET15b vector. b Construction of CrHST into pDEST15 vector

Fig. 3

Scheme of HST catalysis. a Reaction with solanesyl diphosphate as a substrate. b Reaction with farnesyl diphosphate as a substrate

Fig. 4

Purification of CrHST protein. a SDS-PAGE of CrHST using DPC and DDM to purify. Lane1 CrHST in DPC and lane 2 CrHST in DDM. b Homogeneity of CrHST in different detergents analyzed by gel filtration. c CrHST was purified with the Ni²⁺-NTA column. SDS-PAGE determination from chromatograms refers to M, marker; FT, flow-through; W, washed with 10 mM imidazole; and E, elution with 300 mM imidazole. d CrHST was purified with a size-exclusion chromatography column. SDS-PAGE showed elution peaks of purified protein. Western blot for protein identification

Fig. 5

Enzymatic activity assay. a Michaelis constant of HGA to CrHST. b Michaelis constant of FPP to CrHST. c Determination of the IC₅₀ of HST inhibitor DMC

Fig. 6

Sequence analysis of HST and activity assay. a CrHST, AtHST and DGGGPase sequence alignment. b Alignment of conserved sequence motifs. The key residues of two Asp-rich motifs are highlighted in red. c CrHST is a transmembrane protein with 7 predicted transmembrane helices. The transmembrane domain is labeled. d The enzyme activity assay of CrHST mutants. e The putative activity pocket of CrHST

Fig. 7

Proposed binding modes of CrHST with substrate and inhibitor. a Possible binding pocket of CrHST. b Proposed binding modes of CrHST with HGA. c Proposed binding modes of CrHST with DMC. CrHST protein was shown as green cartoon, and the interacting residues were highlighted as stick style. The compounds HGA and DMC were shown as cyan and magenta sticks, respectively