Journal Article

Expression, purification, and characterization of transmembrane protein homogentisate solanesyltransferase

Xiao, Han, Chen, Zhao, Yang, Guang-Fu, Yu, Xin-He, Yang, Jun, Mei, Long-Can, Lin, Hong-Yan, Tong, Qiong

2024

Overview

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

• Publisher: Springer Berlin Heidelberg,Springer,Springer Nature B.V
• Subject: Affinity / Amino acid sequence / Amino Acids / biochemical pathways / Biomedical and Life Sciences / Biosynthesis / Biotechnologically Relevant Enzymes and Proteins / Biotechnology / Chlamydomonas reinhardtii / Chlamydomonas reinhardtii - genetics / Conserved sequence / E coli / energy / Enzymes / Escherichia coli / Escherichia coli - genetics / Herbicides / Herbicides - pharmacology / Life Sciences / Membrane Proteins / Microbial Genetics and Genomics / Microbiology / Molecular docking / Molecular Docking Simulation / Nucleotide sequence / Phenylacetates / Physiological aspects / plastoquinones / Protein purification / Proteins / sequence alignment / Substrate inhibition / Substrate preferences / Testing / Three dimensional analysis / Transferases / Transmembrane proteins