Conserved Amino Acid Residues Research Articles

Vimentin as a universal receptor for pseudorabies virus infection in pig and human cells.

Pseudorabies virus (PRV), known to infect pigs and found in various species, including humans, shows zoonotic potential. This study identified vimentin (VIM), a highly conserved intermediate filament protein expressed in multiple mammalian species and tissues, as a universal receptor for PRV infections in human and porcine cells. The adsorption of PRV is positively correlated with the level of VIM expressed in different cells. Overexpression and knockdown of VIM significantly increase and decrease PRV adsorption in cells, respectively. Dot blot assay and VOPBA showed that purified VIM can directly bind to PRV virions efficiently in a dose-dependent manner. PRV and VIM are co-localized at the cell membrane of PRV-infected cells. Moreover, PRV mainly binds to host VIM via its conserved amino acid residues in PRV gD (W98, G162, Y164, C205) and gH (C439) and the Rod domain (residues 96-404) of VIM. In addition, regulating the expression of VIM also influences the entry, replication, and release of PRV, which has a similar result to the adsorption. These results demonstrate that VIM, as a universal receptor, can facilitate PRV infection in multiple stages in human and porcine cells, highlighting the zoonosis characteristics of PRV and the need for more attention.

Low nucleotide diversity of the Plasmodium falciparum AP2-EXP2 gene among clinical samples from Ghana.

PfAP2-EXP2 is located within chromosome 6 of Plasmodium falciparum recently identified to be undergoing an extensive selective sweep in West African isolates. The gene encoding this transcription factor, PfAP2-EXP2, is essential and thus likely subject to purifying selection that limits variants in the parasite population despite its genomic location. 72 Plasmodium falciparum field samples and 801 clinical sequences from the Pf6 MalariaGEN dataset of Ghanaian origin, were integrated and analysed. A total of 14 single nucleotide variants of which 5 were missense variants, were identified after quality checks and filtering. Except for one, all identified variants were rare among the clinical samples obtained in this study (Minor allelic frequency < 0.01). Further results revealed a considerably low dN/dS value (0.208) suggesting the presence of purifying selection. Further, all the mutant amino acids were wildtype residues in AP2-EXP2 orthologous proteins-tentatively suggesting a genus-level conservation of amino acid residues. Computational analysis and predictions corroborated these findings. Despite the recent extensive selective sweep within chromosome 6 of West African isolates, PfAP2-EXP2 of Ghanaian origin exhibits low nucleotide diversity and very low dN/dS consistent with purifying selection acting to maintain the function of an essential gene. The conservation of AP2-EXP2 is an important factor that makes it a potential drug target.

Cysteine-Persulfide Sulfane Sulfur-Ligated Zn Complex of Sulfur-Carrying SufU in the SufCDSUB System for Fe-S Cluster Biosynthesis.

SufU, a component of the SufCDSUB Fe-S cluster biosynthetic system, serves as a Zn-dependent sulfur-carrying protein that delivers inorganic sulfur in the form of cysteine persulfide from SufS to SufBCD. To understand this sulfur delivery mechanism, we studied the X-ray crystal structure of SufU and its sulfur-carrying state (persulfurated SufU) and performed functional analysis of the conserved amino acid residues around the Zn sites. Interestingly, sulfur-carrying SufU with Cys41-persulfide (Cys41-Sγ-Sδ-) exhibited a unique Zn coordination structure, in which electrophilic Sγ is ligated to Zn and nucleophilic/anionic Sδ is bound to distally conserved Arg125. This structure is distinct from those of other Cys-persulfide-Sδ-ligated metals of metalloproteins, such as hybrid cluster proteins and SoxAX. Functional analysis of SufU variants with Zn-ligand and Arg125 substitutions revealed that both Zn and Arg125 are critical for the function of SufU with SufS. The Zn-persulfide structure of SufU provides insight into the sulfur-transfer process, suggesting that persulfide-Sδ- is stabilized via bridging by Zn and Arg125 of SufU.

Cryptosporidium PI(4)K inhibitor EDI048 is a gut-restricted parasiticidal agent to treat paediatric enteric cryptosporidiosis

Diarrhoeal disease caused by Cryptosporidium is a major cause of morbidity and mortality in young and malnourished children from low- and middle-income countries, with no vaccine or effective treatment. Here we describe the discovery of EDI048, a Cryptosporidium PI(4)K inhibitor, designed to be active at the infection site in the gastrointestinal tract and undergo rapid metabolism in the liver. By using mutational analysis and crystal structure, we show that EDI048 binds to highly conserved amino acid residues in the ATP-binding site. EDI048 is orally efficacious in an immunocompromised mouse model despite negligible circulating concentrations, thus demonstrating that gastrointestinal exposure is necessary and sufficient for efficacy. In neonatal calves, a clinical model of cryptosporidiosis, EDI048 treatment resulted in rapid resolution of diarrhoea and significant reduction in faecal oocyst shedding. Safety and pharmacological studies demonstrated predictable metabolism and low systemic exposure of EDI048, providing a substantial safety margin required for a paediatric indication. EDI048 is a promising clinical candidate for the treatment of life-threatening paediatric cryptosporidiosis.

Computational Analyses of Amino Acid Molecules in PDE7A for Elucidating their Evolutionary Diversity and Protein Interactions in Multiple Mammalian Species

This study explores the protein interactions and evolutionary conservation of phosphodiesterase type 7A (PDE7A) across different species, to identify the species with the highest similarity to humans, which has important applications in many fields such as pharmacology, comparative biology, biochemistry, oncology, endocrinology, and reproductive sciences. Computational analyses of the amino acid sequence of this enzyme revealed a common evolutionary relationship between the selected species and humans, but camels show the most variable evolutionary lineage in their PDE7A protein over time. In searches for homologous proteins, identity analysis results for four returned amino acid sequences were as follows: 91.81 ± 4.25% (mean ± SD), while the similarity between the sequences was 94.66% ± 4.55, and analysis of BLOSUM62 (Blocks Substitution Matrix 62) yielded a minimum value of 0.901 ± 0.055. All species have closely related physicochemical properties. The computational analyses revealed conserved amino acid residues of the gene across the studied species, which likely contribute to the similar expression patterns of the PDE7A gene in various animals. The findings also suggest that rats are a suitable model for gaining deeper insights into human biology.

Genome-wide analysis and prediction of chloroplast and mitochondrial RNA editing sites of AGC gene family in cotton (Gossypium hirsutum L.) for abiotic stress tolerance

BackgroundCotton is one of the topmost fiber crops throughout the globe. During the last decade, abrupt changes in the climate resulted in drought, heat, and salinity. These stresses have seriously affected cotton production and significant losses all over the textile industry. The GhAGC kinase, a subfamily of AGC group and member of serine/threonine (Ser/Thr) protein kinases group and is highly conserved among eukaryotic organisms. The AGC kinases are compulsory elements of cell development, metabolic processes, and cell death in mammalian systems. The investigation of RNA editing sites within the organelle genomes of multicellular vascular plants, such as Gossypium hirsutum holds significant importance in understanding the regulation of gene expression at the post-transcriptional level.MethodsIn present work, we characterized twenty-eight GhAGC genes in cotton and constructed phylogenetic tree using nine different species from the most primitive to the most recent.ResultsIn sequence logos analyses, highly conserved amino acid residues were found in G. hirsutum, G. arboretum, G. raimondii and A. thaliana. The occurrence of cis-acting growth and stress-related elements in the promoter regions of GhAGCs highlight the significance of these factors in plant development and abiotic stress tolerance. Ka/Ks levels demonstrated that purifying selection pressure resulting from segmental events was applied to GhAGC with little functional divergence. We focused on identifying RNA editing sites in G. hirsutum organelles, specifically in the chloroplast and mitochondria, across all 28 AGC genes.ConclusionThe positive role of GhAGCs was explored by quantifying the expression in the plant tissues under abiotic stress. These findings help in understanding the role of GhAGC genes under abiotic stresses which may further be used in cotton breeding for the development of climate smart varieties in abruptly changing climate.

Subtranscriptome analysis of phospholipases D in Loxosceles venom glands: Confirmation of predominance, intra-species diversity, and description of novel isoforms

Spiders of Loxosceles genus, or Brown spiders produce a potent venom with minimal volume and protein content. Among its toxins, phospholipases D (PLDs) are notable for causing primary local and systemic manifestations observed following envenomation. They degrade cellular phospholipids, mainly sphingomyelin and lysophosphatidylcholine. We present a robust and detailed analysis of PLD transcripts from venom glands of three major clinically relevant South American species—L. intermedia, L. laeta, and L. gaucho—using next-generation sequencing. Results confirmed that PLDs are the most highly expressed toxins, accounting for 65.4 % of expression in L. intermedia, 71.8 % in L. gaucho, and 50.4 % in L. laeta. These findings further support the idea that these enzymes form a protein family both within and across species. Eighteen contigs for PLDs were found for L. gaucho, 24 for L. intermedia, and 21 for L. laeta. A detailed analysis revealed that, although all contigs display conserved amino acid residues directly involved in catalysis, magnesium coordination, and substrate affinity, they also possess distinct primary sequences with important substitutions. Such data reinforces the hypothesis that these toxins may act synergistically. Furthermore, new PLD sequences were identified within the contigs. For L. intermedia, 14 potential new isoforms were identified; 16 for L gaucho; and 16 novel sequences for L. laeta. This indicates that there is still a wealth of undisclosed information about these toxins. These data will help identify structural and functional differences among these proteins, support future functional studies, and to the comprehensive understanding of the mechanism of action of PLDs.

Enhancing the imidase activity of BpIH toward 3-isobutyl glutarimide via semi-rational design

(R)-3-Isobutylglutarate monoamide (R-IBM) is a key intermediate in the synthesis of the analgesic drug pregabalin. Recently, the imidase BpIH derived from Burkholderia phytofirmans was identified as a promising catalyst for the industrial production of R-IBM. Notably, this catalyst has the distinct advantage of achieving a 100% theoretical yield from 3-isobutyl glutarimide (IBI). In this study, homology modeling and structure alignment techniques were used to determine the substrate binding pocket of BpIH. Semi-rational design was used to analyze the amino acid residue conservation in the binding pocket region of BpIH. Interestingly, mutations of several low-conserved amino acid located 6–9 Å from the substrate significantly enhanced the catalytic activity of BpIH. Among them, the triple mutant Y37FH133NS226I (YHS-I) showed approximately a fivefold increase in enzyme activity and a significantly improved catalytic efficiency (kcat/Km). Under the same reaction time and conditions, YHS-I successfully converted IBI into R-IBM with a conversion rate of 88.87%, with an enantiomeric excess (ee) of the product exceeding 99.9%. In comparison, wild-type BpIH had a conversion rate of only 38.15%. Molecular dynamics and docking results indicated that YHS-I had higher rigidity around the mutation sites. The synergistic substitutions of Y37F, H133N, and S226I altered the interaction network within the mutation site, enhancing the protein’s affinity for the substrate and improving catalytic efficiency.Key points• 100% theoretical yield of R-IBM by BpIH compared with 50% by resolution• Semi-rational design of BpIH based on conservativity with homologous enzymes• Mutant with enzyme activity of sixfold and product ee value of 99.9%

The structure investigation of GH174 endo-1,3-fucanase revealed an unusual glycoside hydrolase fold

Sulfated fucan has attracted increasing research interest due to its various biological activities. Endo-1,3-fucanases are favorable tools for structure investigation and structure-activity relationships establishment of sulfated fucan. However, the three-dimensional structure of enzymes from the GH174 family has not been disclosed, which hinders the understanding of the action mechanism. This study reports the first crystal structure of endo-1,3-fucanase from GH174 family (Fun174A) at a resolution of 1.60 Å. Notably, Fun174A exhibited an unusual distorted β-sandwich fold, which is distinct from other known glycoside hydrolase folds. The conserved amino acid residues D119 and H154 were proposed as the catalytic residues in the family. Molecular docking suggested that Fun174A primarily recognized sulfated fucan through a series of polar amino acid residues around the substrate binding pocket. Furthermore, structural bioinformatics analysis suggested that the structural analogs of Fun174A may be extensively implicated in the bacterial metabolism of polysaccharides, which provided opportunities for the discovery of novel glycoside hydrolases. This study offers new insights into the structural diversity of glycoside hydrolases and will contribute to the establishment of a novel clan of glycoside hydrolases.

Structural insights and antimicrobial synergy of a proto-galectin from the marine sponge Aiolochroia crassa

In this study, we isolated a novel lectin from the marine sponge Aiolochroia crassa, named AcrL. The lectin showed a preference for glycans containing sialic acid terminal residues, as indicated by the strongest inhibition with fetuin and bovine submaxillary mucin. Primary structure determination by mass spectrometry revealed that AcrL is a galectin with conserved amino acid residues typically involved in carbohydrate binding. Structural modeling indicated that AcrL adopts a typical galectin β-sandwich motif, featuring two anti-parallel β-sheets with five strands each. Docking calculations revealed a carbohydrate-binding site composed of a main site, capable of hosting galactopyranosides, and an extended site, facilitating the binding of complex carbohydrates. AcrL inhibited significant biofilm formation against Staphylococcus aureus, S. epidermidis, and Escherichia coli with concentrations ranging from 500 to 15.6 μg.mL−1 for S. aureus, 7.8 μg.mL−1 for S. epidermidis, and 500 μg.mL−1 for E. coli. Furthermore, when combined with different antibiotics, AcrL potentiated their effect against pathogenic bacteria. The antimicrobial mechanism of AcrL was investigated using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The analysis indicates that AcrL induces damage to the bacterial membrane. These findings underscore the discovery of a novel galectin in a basal organism and the comprehensive biochemical characterization conducted in this research, highlighting the potential of AcrL as a novel antibacterial agent and emphasizing its importance in combating bacterial infections.

Prediction of antibiotic resistance mechanisms using a protein language model.

Antibiotic resistance has emerged as a major global health threat, with an increasing number of bacterial infections becoming difficult to treat. Predicting the underlying resistance mechanisms of antibiotic resistance genes (ARGs) is crucial for understanding and combating this problem. However, existing methods struggle to accurately predict resistance mechanisms for ARGs with low similarity to known sequences and lack sufficient interpretability of the prediction models. In this study, we present a novel approach for predicting ARG resistance mechanisms using ProteinBERT, a protein language model (pLM) based on deep learning. Our method outperforms state-of-the-art techniques on diverse ARG datasets, including those with low homology to the training data, highlighting its potential for predicting the resistance mechanisms of unknown ARGs. Attention analysis of the model reveals that it considers biologically relevant features, such as conserved amino acid residues and antibiotic target binding sites, when making predictions. These findings provide valuable insights into the molecular basis of antibiotic resistance and demonstrate the interpretability of pLMs, offering a new perspective on their application in bioinformatics. The source code is available for free at https://github.com/hmdlab/ARG-BERT. The output results of the model are published at https://waseda.box.com/v/ARG-BERT-suppl.

Molecular Characterization, Evolution and Expression Analysis of Ammonium Transporter from Four Closely Related Bactrocera Species (Tephritidae).

Numerous insects are attracted to low levels of ammonia, utilizing it as a cue to locate food sources. The Ammonium Transporter (Amt), a highly conserved, atypical olfactory receptor, has been shown to mediate the detection of ammonia in insects. While the attraction of Tephritidae to ammonia is well established, knowledge about the Amt in this family is limited. The species Bactrocera dorsalis (Hendel 1912), Bactrocera cucurbitae (Coquillett 1899), Bactrocera correcta Bezzi 1916 and Bactrocera tau (Walker 1849), which are common agricultural pests within Tephritidae, exhibit numerous ecological similarities, offering a solid foundation for studying Amt characteristics in this family. In this study, we elucidated the sequences, evolutionary relationships, and expression patterns of Amt in these four species. The results indicated that these Amts share the same open reading frame, containing 1770 bp that encode a protein of 589 amino acid residues. These Amt proteins exhibit the typical structural characteristics of Amts, including an 11-transmembrane domain with an extracellular N-terminus and an intracellular C-terminus. They also have the ability to form trimers in the membrane. Additionally, they contain three conserved amino acid residues essential for ammonia transport: A189, H195, and H352. Phylogenetic and expression pattern analyses showed that they are highly conserved in Diptera and are significantly expressed in antennae. This study is the first report characterizing the Amt gene in four Tephritidae species. These findings provide a foundation for further exploration into the roles of these genes in their particular biological contexts.

De Novo Missense Variations of ATP8B2 Impair Its Phosphatidylcholine Flippase Activity.

P4-ATPases comprise a family of lipid flippases that translocate lipids from the exoplasmic (or luminal) to the cytoplasmic leaflet of biological membranes. Of the 14 known human P4-ATPases, ATP8B2 is a phosphatidylcholine flippase at the plasma membrane, but its physiological function is not well understood. Although ATP8B2 could interact with both CDC50A and CDC50B, it required only the CDC50A interaction for its exit from the endoplasmic reticulum and subsequent transport to the plasma membrane. Three de novo monoallelic missense variations of ATP8B2 were found in patients with intellectual disability. None of these variations affected the interaction of ATP8B2 with CDC50A or its localization to the plasma membrane. However, variations of either of two amino acid residues, which are conserved in all P4-ATPases, significantly reduced the phosphatidylcholine flippase activity of ATP8B2. Furthermore, mutations in the corresponding residues of ATP8B1 and ATP11C were found to decrease their flippase activities toward phosphatidylcholine and phosphatidylserine, respectively. These results indicate that the conserved amino acid residues are crucial for the enzymatic activities of the P4-ATPases.

Conserved amino acid residues and gene expression patterns associated with the substrate preferences of the competing enzymes FLS and DFR.

Flavonoids, an important class of specialized metabolites, are synthesized from phenylalanine and present in almost all plant species. Different branches of flavonoid biosynthesis lead to products like flavones, flavonols, anthocyanins, and proanthocyanidins. Dihydroflavonols form the branching point towards the production of non-colored flavonols via flavonol synthase (FLS) and colored anthocyanins via dihydroflavonol 4-reductase (DFR). Despite the wealth of publicly accessible data, there remains a gap in understanding the mechanisms that mitigate competition between FLS and DFR for the shared substrate, dihydroflavonols. An angiosperm-wide comparison of FLS and DFR sequences revealed the amino acids at positions associated with the substrate specificity in both enzymes. A global analysis of the phylogenetic distribution of these amino acid residues revealed that monocots generally possess FLS with Y132 (FLSY) and DFR with N133 (DFRN). In contrast, dicots generally possess FLSH and DFRN, DFRD, and DFRA. DFRA, which restricts substrate preference to dihydrokaempferol, previously believed to be unique to strawberry species, is found to be more widespread in angiosperms and has evolved independently multiple times. Generally, angiosperm FLS appears to prefer dihydrokaempferol, whereas DFR appears to favor dihydroquercetin or dihydromyricetin. Moreover, in the FLS-DFR competition, the dominance of one over the other is observed, with typically only one gene being expressed at any given time. This study illustrates how almost mutually exclusive gene expression and substrate-preference determining residues could mitigate competition between FLS and DFR, delineates the evolution of these enzymes, and provides insights into mechanisms directing the metabolic flux of the flavonoid biosynthesis, with potential implications for ornamental plants and molecular breeding strategies.

Hydrogen-Bonding and Hydrophobic Interaction Networks as Structural Determinants of Microbial Rhodopsin Function.

Microbial pump rhodopsins are highly versatile light-driven membrane proteins that couple protein conformational dynamics with ion translocation across the cell membranes. Understanding how microbial pump rhodopsins use specific amino acid residues at key functional sites to control ion selectivity and ion pumping direction is of general interest for membrane transporters, and could guide site-directed mutagenesis for optogenetics applications. To enable direct comparisons between proteins with different sequences we implement, for the first time, a unique numbering scheme for the microbial pump rhodopsin residues, NS-mrho. We use NS-mrho to show that distinct microbial pump rhodopsins typically have hydrogen-bond networks that are less conserved than anticipated from the amino acid residue conservation, whereas their hydrophobic interaction networks are largely conserved. To illustrate the role of the hydrogen-bond networks as structural elements that determine the functionality of microbial pump rhodopsins, we performed experiments, atomic-level simulations, and hydrogen bond network analyses on GR, the outward proton pump from Gloeobacter violaceus, and KR2, the outward sodium pump from Krokinobacter eikastus. The experiments indicate that multiple mutations that recover KR2 amino acid residues in GR not only fail to convert it into a sodium pump, but completely inactivate GR by abolishing photoisomerization of the retinal chromophore. This observation could be attributed to the drastically altered hydrogen-bond interaction network identified with simulations and network analyses. Taken together, our findings suggest that functional specificity could be encoded in the collective hydrogen-bond network of microbial pump rhodopsins.

Novel WFS1 Variants in Two Moroccan Families with Wolfram Syndrome.

Background: Wolfram syndrome (WFS) is an autosomal recessive disorder that often leads to diabetes, optic atrophy, and sensorineural hearing loss. The aim of this study was to determine the clinical characteristics and the genetic cause of the first two Moroccan families presenting with WFS. Methods: The clinical features of five members of two WFS families were evaluated. Whole-exome sequencing was conducted to explore the underlying genetic cause in the affected patients. Results: Two homozygous variants in the WFS1 gene were identified, each in one of the two families studied: a missense c.1329C>G variant (p.Ser443Arg) and a nonsense mutation c.1113G>A (p.Trp371Ter). These variants affected conserved amino acid residues, segregated well in the two families, and are absent from genetic databases and in controls of Moroccan origin. Bioinformatics analysis classified the two variants as pathogenic by in silico tools and molecular modeling. Conclusion: Our study identified for the first time two variants in Moroccan patients with WFS that extends the mutational spectrum associated with the disease.

Genetic analysis of seven patients with inherited ichthyosis and Nagashima‑type palmoplantar keratoderma.

Inherited ichthyosis comprises a series of heterogeneous dermal conditions; it mainly manifests as widespread hyperkeratosis, xerosis and scaling of the skin. At times, overlapping symptoms require differential diagnosis between ichthyosis and several other similar disorders. The present study reports seven patients with confirmed or suspected to be associated with ichthyosis by conducting a thorough clinical and genetic investigation. Genetic testing was conducted using whole‑exome sequencing, with Sanger sequencing as the validation method. The MEGA7 program was used to analyze the conservation of amino acid residues affected by the detected missense variants. The enrolled patients exhibited ichthyosis‑like but distinct clinical manifestations. Genetic analysis identified diagnostic variations in the FLG, STS, KRT10 and SERPINB7 genes and clarified the carrying status of each variant in the respective family members. The two residues affected by the detected missense variants remained conserved across multiple species. Of note, the two variants, namely STS: c.452C>T(p.P151L) and c.647_650del(p.L216fs) are novel. In conclusion, a clear genetic differential diagnosis was made for the enrolled ichthyosis‑associated patients; the study findings also extended the mutation spectrum of ichthyosis and provided solid evidence for the counseling of the affected families.

Marein, a novel natural product for restoring chemo-sensitivity to cancer cells through competitive inhibition of ABCG2 function

The pivotal roles of ATP-binding cassette (ABC) transporters in drug resistance have been widely appreciated. Here we report that marein, a natural product from Coreopsis tinctoria Nutt, is a potent chemo-sensitizer in drug resistant cancer cells overexpressing ABCG2 transporter. We demonstrate that marein can competitively inhibit efflux activity of ABCG2 protein and increase the intracellular accumulation of the chemotherapeutic drugs that belong to substrate of this transporter. We further show that marein can bind to the conserved amino acid residue F439 of ABCG2, a critical site for drug-substrate interaction. Moreover, marein can significantly sensitize the ABCG2-expressing tumor cells to chemotherapeutic drugs such as topotecan, mitoxantrone, and olaparib. This study reveals a novel role and mechanism of marein in modulating drug resistance, and may have important implications in treatment of cancers that are resistant to chemotherapeutic drugs that belong to the substrates of ABCG2 transporters.

Conserved proline residues prevent dimerization and aggregation in the β-lactamase BlaC.

Evolution leads to conservation of amino acid residues in protein families. Conserved proline residues are usually considered to ensure the correct folding and to stabilize the three-dimensional structure. Surprisingly, proline residues that are highly conserved in class A β-lactamases were found to tolerate various substitutions without large losses in enzyme activity. We investigated the roles of three conserved prolines at positions 107, 226, and 258 in the β-lactamase BlaC from Mycobacterium tuberculosis and found that mutations can lead to dimerization of the enzyme and an overall less stable protein that is prone to aggregate over time. For the variant Pro107Thr, the crystal structure shows dimer formation resembling domain swapping. It is concluded that the proline substitutions loosen the structure, enhancing multimerization. Even though the enzyme does not lose its properties without the conserved proline residues, the prolines ensure the long-term structural integrity of the enzyme.

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 (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 Km 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