Construction of carotenoid biosynthetic pathways using squalene synthase

The first committed steps of steroid/hopanoid pathways involve squalene synthase (SQS). Here, we report the Escherichia coli production of diaponeurosporene and diapolycopene, yellow C₃₀ carotenoid pigments, by expressing human SQS and Staphylococcus aureus dehydrosqualene (C₃₀ carotenoid) desaturase (CrtN). We suggest that the carotenoid pigments are synthesized mainly via the desaturation of squalene rather than the direct synthesis of dehydrosqualene through the non-reductive condensation of prenyl diphosphate precursors, indicating the possible existence of a “squalene route” and a “lycopersene route” for C₃₀ and C₄₀ carotenoids, respectively. Additionally, this finding yields a new method of colorimetric screening for the cellular activity of squalene synthases, which are major targets for cholesterol-lowering drugs.     

In silico analysis of squalene synthase in Fabaceae family using bioinformatics tools

Triterpenoid saponins are a diverse group of bioactive compounds, which are used for possessing of many biomedical and pharmaceutical products. Generally, squalene synthase (SQS) is defined as an emerging and essential branch point enzyme far from the major pathway of isoprenoids biosynthetic and a latent adjusting point, which manages carbon flux into triterpenes biosynthesis and sterols. The present study deals with the detailed characterization of SQS by bioinformatics approaches to evaluate physicochemical properties, structural characteristics including secondary and 3D structure prediction and functional analysis from eight plants related to Fabaceae family and Arabidopsis thaliana. Bioinformatics analysis revealed that SQS proteins have two transmembrane regions in the C-terminal. The predicted motifs were used to design universal degenerate primers for PCR analysis and other molecular applications. Phylogenetic analysis showed conserved regions at different stretches with maximum homology in amino acid residues within all SQSs. The secondary structure prediction results showed that the amino acid sequence of all squalene synthases had α helix and random coil as the main components. The reliability of the received model was confirmed using the ProSA and RAMPAGE programs. Determining of active site by CASTp proposes the possibility of using this protein as probable medication target. The findings of the present study may be useful for further assessments on characterization and cloning of squalene synthase.     

A novel templates of piperazinyl-1,2-dihydroquinoline-3-carboxylates: Synthesis,anti-microbial evaluation and molecular docking studies

A series of piperazinyl-1,2-dihydroquinoline carboxylates were synthesized by the reaction of ethyl 4-chloro-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylates with various piperazines and their structures were confirmed by ¹H NMR, ¹³C NMR, IR and mass spectral analysis. All the synthesized compounds were screened for their in vitro antimicrobial activities. Further, the in silico molecular docking studies of the active compounds was performed to explore the binding interactions between piperazinyl-1,2-dihydroquinoline carboxylate derivatives and the active site of the Staphylococcus aureus (CrtM) dehydrosqualene synthase (PDB ID: 2ZCQ). The docking studies revealed that the synthesized derivatives showed high binding energies and strong H-bond interactions with the dehydrosqualene synthase validating the observed antimicrobial activity data. Based on antimicrobial activity and docking studies, the compounds 9b and 10c were identified as promising antimicrobial lead molecules. This study might provide insights to identify new drug candidates that target the S. aureus virulence factor, dehydrosqualene synthase.     

Molecular cloning and expression analysis of a squalene synthase gene from a medicinal plant, Euphorbia pekinensis Rupr.

Euphorbia pekinensis Rupr., which is also known as a medicinal plant, produces a large amount of alkaloids, phytosterols and triterpenes. In this study, we reported on the cDNA cloning and characterization of a novel squalene synthase (SQS) from E. pekinensis. Squalene synthase catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to produce squalene (SQ), the first committed precursor for sterol and triterpene biosynthesis. The full length cDNA named EpSQS (Genbank Accession Number JX509735) contained 1,614 bp with an open reading frame of 1,236 bp encoding a polypeptide of 411 amino acids. The deduced amino acid sequence of the EpSQS named EpSQS exhibited a high homology with other plant SQSs, and contained a single domain surrounded by helices. Phylogenetic analysis showed that EpSQS belonged to the plant SQS kingdom. Tissue expression analysis revealed that EpSQS expressed strongly in roots, weakly in stems and leaves, implying that EpSQS was a constitutive expression gene. The recombinant protein was expressed in Escherichia coli and detected by SDS-PAGE and western blot. The high performance liquid chromatography (HPLC) analysis showed that EpSQS could catalyze the reaction from farnesyl diphosphate (FPP) to squalene.     

Cloning and Functional Characterization of a Squalene Synthase from Paris polyphylla var. yunnanensis

Paris polyphylla Smith var. yunnanensis (Franch.) Hand. – Mazz. is a precious traditional Chinese medicine, and steroidal saponins are its major bioactive constituents possessing extensive biological activities. Squalene synthase (SQS) catalyzes the first dedicated step converting two molecular of farnesyl diphosphate (FDP) into squalene, a key intermediate in the biosynthetic pathway of steroidal saponins. In this study, a squalene synthase gene (PpSQS1) was cloned and functionally characterized from P. polyphylla var. yunnanensis, representing the first identified SQS from the genus Paris. The open reading frame of PpSQS1 is 1239 bp, which encodes a protein of 412 amino acids showing high similarity to those of other plant SQSs. Expression of PpSQS1 in Escherichia coli resulted in production of soluble recombinant proteins. Gas chromatography-mass spectrometry analysis showed that the purified recombinant PpSQS1 protein could produce squalene using FDP as a substrate in the in vitro enzymatic assay. qRT-PCR analysis indicated that PpSQS1 was highly expressed in rhizomes, consistent with the dominant accumulation of steroidal saponins there, suggesting that PpSQS1 is likely involved in the biosynthesis of steroidal saponins in the plant. The findings lay a foundation for further investigation on the biosynthesis and regulation of steroidal saponins, and also provide an alternative gene for manipulation of steroid production using synthetic biology.     

Potential antimicrobial agents from triazole-functionalized 2H-benzo[b][1,4]oxazin-3(4H)-ones

A series of substituted triazole functionalized 2H-benzo[b][1,4]oxazin-3(4H)-ones were synthesized by employing click chemistry and further characterized based on ¹H NMR, ¹³C NMR, IR and mass spectral studies. All the synthesized derivatives were screened for their in vitro antimicrobial activities. Further, molecular docking studies were accomplished to explore the binding interactions between 1,2,3-triazol-4-yl-2H-benzo[b][1,4]oxazin-3(4H)-one and the active site of Staphylococcus aureus (CrtM) dehydrosqualene synthase (PDB ID: 2ZCS). These docking studies revealed that the synthesized derivatives showed high binding energies and strong H-bond interactions with the dehydrosqualene synthase validating the observed antimicrobial activity data. Based on antimicrobial activity and docking studies, the compounds 9c, 9d and 9e were identified as promising antimicrobial leads.     

Archaeal Pus10 proteins can produce both pseudouridine 54 and 55 in tRNA

Pus10, a recently identified pseudouridine (Ψ) synthase, does not belong to any of the five commonly identified families of Ψ synthases. Pyrococcus furiosus Pus10 has been shown to produce Ψ55 in tRNAs. However, in vitro studies have identified another mechanism for tRNA Ψ55 production in Archaea, which uses Cbf5 and other core proteins of the H/ACA ribonucleoprotein complex, in a guide RNA-independent manner. Pus10 homologs have been observed in nearly all sequenced archaeal genomes and in some higher eukaryotes, but not in yeast and bacteria. This coincides with the presence of Ψ54 in the tRNAs of Archaea and higher eukaryotes and its absence in yeast and bacteria. No tRNA Ψ54 synthase has been reported so far. Here, using recombinant Methanocaldococcus jannaschii and P. furiosus Pus10, we show that these proteins can function as synthase for both tRNA Ψ54 and Ψ55. The two modifications seem to occur independently. Salt concentration dependent variations in these activities of both proteins are observed. The Ψ54 synthase activity of M. jannaschii protein is robust, while the same activity of P. furiosus protein is weak. Probable reasons for these differences are discussed. Furthermore, unlike bacterial TruB and yeast Pus4, archaeal Pus10 does not require a U54•A58 reverse Hoogstein base pair and pyrimidine at position 56 to convert tRNA U55 to Ψ55. The homology of eukaryal Pus10 with archaeal Pus10 suggests that the former may also have a tRNA Ψ54 synthase activity.     

Cloning, expression and characterization of squalene synthase from Inonotus obliquus

The chaga mushroom Inonotus obliquus has been widely used as a folk medicine in Russia, Poland and most of the Baltic countries. The total triterpene saponins of I. obliquus have significant pharmacological activity. Though the triterpene component has been well characterized in terms of its pharmaceutical activity, there is little information on the genes responsible for the biosynthesis of these compounds in I. obliquus. Squalene synthase represents a potential branching point and the first committed step to diverge the carbon flux from the main isoprenoid pathway towards sterol biosynthesis. In this study, we cloned and characterized squalene synthase from I. obliquus. A 1476-bp full-length cDNA consisting of the entire coding region of squalene synthase (GenBank accession number is KC182754) was cloned by RT-PCR. The DNA sequence showed as much as 76 % similarity with the sequence of Fomitiporia mediterranea squalene synthase, and phylogenetic analysis indicated that it is most closely related to F. mediterranea squalene synthase at both DNA and protein levels. I. obliquus squalene synthase was actively expressed in the yeast Pichia pastoris as a secreted form and purified by gel filtration using Superdex G-75 column. The purified recombinant squalene synthase was able to convert farnesyl diphosphate (FPP) to squalene in an NADPH-dependent reaction. The result of this study could serve as an important step toward the manipulation of triterpenoids biosynthesis in I. obliquus at the level of squalene through engineering better SQS for reintroduction into the mushroom.     

Binding modes of zaragozic acid A to human squalene synthase and staphylococcal dehydrosqualene synthase

Zaragozic acids (ZAs) belong to a family of fungal metabolites with nanomolar inhibitory activity toward squalene synthase (SQS). The enzyme catalyzes the committed step of sterol synthesis and has attracted attention as a potential target for antilipogenic and antiinfective therapies. Here, we have determined the structure of ZA-A complexed with human SQS. ZA-A binding induces a local conformational change in the substrate binding site, and its C-6 acyl group also extends over to the cofactor binding cavity. In addition, ZA-A effectively inhibits a homologous bacterial enzyme, dehydrosqualene synthase (CrtM), which synthesizes the precursor of staphyloxanthin in Staphylococcus aureus to cope with oxidative stress. Size reduction at Tyr(248) in CrtM further increases the ZA-A binding affinity, and it reveals a similar overall inhibitor binding mode to that of human SQS/ZA-A except for the C-6 acyl group. These structures pave the way for further improving selectivity and development of a new generation of anticholesterolemic and antimicrobial inhibitors.     

Design,synthesis, neuroprotective,antibacterial activities and docking studies of novel thieno[2,3-d]pyrimidine-alkyne Mannich base and oxadiazole hybrids

A series of thieno[2,3-d]pyrimidine alkyne Mannich base derivatives (7a-e, 8a-e) and thieno[2,3-d]pyrimidine 1,3,4-oxadiazole derivatives (9a-e, 10a-e) have been synthesized and evaluated for their neuroprotective and neurotoxicity activities where 9a, 10d displayed good neuroprotection 10.6 and 11.88?µg/mL respectively against the H₂O₂ induced cell death at the EC₅₀ values and 9b, 9d showed respective toxic effects on PC12 cells at CC₅₀ 86.12 and 94.16?µg/mL. Compounds 9a, 9e, 10a and 10b showed strong antibacterial activity against two gram positive (S. aureus, B. subtilis) and two gram-negative strains (E. coli, P. aeruginosa) and showed good binding affinities with C(30) carotenoid dehydrosqualene synthase, Gyrase A and LpxC. This is the first report for the demonstration of thieno[2,3-d] pyrimidine derivatives as promising neuroprotective agents against H₂O₂ induced neurotoxicity on PC12 cells.     

Single‐molecule real‐time sequencing reveals diverse allelic variations in carotenoid biosynthetic genes in pepper (Capsicum spp.)

The diverse colours of mature pepper (Capsicum spp.) fruit result from the accumulation of different carotenoids. The carotenoid biosynthetic pathway has been well elucidated in Solanaceous plants, and analysis of candidate genes involved in this process has revealed variations in carotenoid biosynthetic genes in Capsicum spp. However, the allelic variations revealed by previous studies could not fully explain the variation in fruit colour in Capsicum spp. due to technical difficulties in detecting allelic variation in multiple candidate genes in numerous samples. In this study, we uncovered allelic variations in six carotenoid biosynthetic genes, including phytoene synthase (PSY1, PSY2), lycopene β‐cyclase, β‐carotene hydroxylase, zeaxanthin epoxidase and capsanthin‐capsorubin synthase (CCS) genes, in 94 pepper accessions by single‐molecule real‐time (SMRT) sequencing. To investigate the relationship between allelic variations in the candidate genes and differences in fruit colour, we performed ultra‐performance liquid chromatography analysis using 43 accessions representing each allelic variation. Different combinations of dysfunctional mutations in PSY1 and CCS could explain variation in the compositions and levels of carotenoids in the accessions examined in this study. Our results demonstrate that SMRT sequencing technology can be used to rapidly identify allelic variation in target genes in various germplasms. The newly identified allelic variants will be useful for pepper breeding and for further analysis of carotenoid biosynthesis pathways.     

Squalene Synthase As a Target for Chagas Disease Therapeutics

Trypanosomatid parasites are the causative agents of many neglected tropical diseases and there is currently considerable interest in targeting endogenous sterol biosynthesis in these organisms as a route to the development of novel anti-infective drugs. Here, we report the first x-ray crystallographic structures of the enzyme squalene synthase (SQS) from a trypanosomatid parasite, Trypanosoma cruzi, the causative agent of Chagas disease. We obtained five structures of T. cruzi SQS and eight structures of human SQS with four classes of inhibitors: the substrate-analog S-thiolo-farnesyl diphosphate, the quinuclidines E5700 and ER119884, several lipophilic bisphosphonates, and the thiocyanate WC-9, with the structures of the two very potent quinuclidines suggesting strategies for selective inhibitor development. We also show that the lipophilic bisphosphonates have low nM activity against T. cruzi and inhibit endogenous sterol biosynthesis and that E5700 acts synergistically with the azole drug, posaconazole. The determination of the structures of trypanosomatid and human SQS enzymes with a diverse set of inhibitors active in cells provides insights into SQS inhibition, of interest in the context of the development of drugs against Chagas disease.     

Molecular cloning,prokaryotic expression and promoter analysis of squalene synthase gene from Schizochytrium Limacinum

Squalene synthase (SQS) is an important enzyme in the steroid biosynthetic pathways which condenses two molecules of farnesyl pyrophosphate into a squalene. In this study, the gene encoding SQS was isolated from Schizochytrium limacinum and characterized. The full-length cDNA of S. limacinum SQS gene (SlSQS) is 1605 bp in length, it contains a 1293 bp ORF encoding a polypeptide of 430 amino acids. Multiple amino acid sequence alignment showed that the SlSQS protein sequence shared 5 conserved signature domains and a hydrophobic carboxy-terminal part with other known SQS protein sequences. C-terminal-truncated SlSQS was constructed into expression vector pGEX and successfully expressed in Escherichia coli cells. The expressed fusion protein was confirmed to have SQS activity. In addition, a 724 bp promoter region of SlSQS was also cloned and several cis-acting elements were predicted. These results might be helpful to understand the structure and expression regulation of SQS in S. limacinum.     

Carotenoid biosynthesis and the evolution of carotenogenesis genes in rust fungi

Diseases caused by rust fungi pose a significant threat to global plant production. Although carotenoid pigments are produced in spores of nearly all rust species, the corresponding biosynthesis pathway(s) have not been investigated. Here, candidate genes for carotenoid biosynthesis in Puccinia graminis f. sp. tritici (Pgt) were identified, cloned and functionally complemented using specifically engineered strains of Escherichia coli. A part of the carotenoid biosynthesis pathway in rust fungi was elucidated, with only two genes, CrtYB and CrtI, catalysing the reactions from geranyl–geranyl diphosphate (GGPP) to γ-carotene. The CrtYB gene encodes a bi-functional lycopene cyclase/phytoene synthase, which catalyses the condensation of two GGPP into phytoene, as well as the cyclisation of the ψ-end of lycopene to form γ-carotene. The CrtI gene encodes a phytoene desaturase that carries out four successive desaturations of phytoene, through the intermediates phytofluene and neurosporene to lycopene. The evolution of carotenoid pigmentation in rust fungi, including Pgt, P. graminis avenae, P. graminis secalis (Pgs), P. graminis lolli, P. striiformis f. sp. tritici, P. striiformis f. sp. pseudohordei, P. striiformis f. sp. hordei, the “scabrum” rust (putative hybrids between Pgt and Pgs), P. triticina, and P. hordei, was investigated by phylogenetic analysis. Both CrtYB and CrtI were found to be closely related among rust fungi, other pathogenic fungi, and some aphids. Our results provide a springboard to increase the understanding of the physiological role(s) of carotenoid pigments in rust fungi, to better understand evolution within the Pucciniales, and to develop robust molecular diagnostics for rust fungi.     

Polish Soil Quality Standards Versus Risk-Based Soil Screening Levels for Metals and Arsenic

The main objective of the study was derivation of risk-based soil screening levels (RBSSLs) under two basic exposure scenarios—industrial and residential, and their comparison with the relevant soil quality standards (SQSs), applied in Poland as remedial targets. The RBSSLs were derived from standardized sets of equations that are based on the recently updated U.S. Environmental Protection Agency's human health risk assessment methods. The article presents the results concerning 12 contaminants listed in the SQS ordinance: arsenic, barium, cadmium, cobalt, chromium, copper, mercury, molybdenum, nickel, lead, tin, and zinc. Taking only the human health protection criterion into account, most of SQSs for non-carcinogenic metals under both industrial and residential scenarios seem to be too stringent if used as the remedial levels, which may lead to unnecessary remediation. On the other hand, the SQSs for carcinogenic contaminants (As, Cr^VI) correspond to cancer risk levels significantly higher than the acceptable level of 1E-06. The findings of the study may constitute the first step to justify the amendment of the Polish SQS ordinance aimed at establishing the new soil quality values based on clearly defined criteria.     

Expression and characterization of PhzE from P. aeruginosa PAO1: aminodeoxyisochorismate synthase involved in pyocyanin and phenazine-1-carboxylate production

PhzE from Pseudomonas aeruginosa catalyzes the first step in the biosynthesis of phenazine-1-carboxylic acid, pyocyanin, and other phenazines, which are virulence factors for Pseudomonas species. The reaction catalyzed converts chorismate into aminodeoxyisochorismate using ammonia supplied by a glutamine amidotransferase domain. It has structural and sequence homology to other chorismate-utilizing enzymes such as anthranilate synthase, isochorismate synthase, aminodeoxychorismate synthase, and salicylate synthase. Like these enzymes, it is Mg^2 + dependent and catalyzes a similar S_N2" nucleophilic substitution reaction. PhzE catalyzes the addition of ammonia to C2 of chorismate, as does anthranilate synthase, yet unlike anthranilate synthase it does not catalyze elimination of pyruvate from enzyme-bound aminodeoxyisochorismate. Herein, the cloning of the phzE gene, high level expression of active enzyme in E. coli, purification, and kinetic characterization of the enzyme is presented, including temperature and pH dependence. Steady-state kinetics give K_chorismate = 20 ± 4 μM, K_Mg^2 + = 294 ± 22 μM, K_L-gln = 11 ± 1 mM, and k_cat = 2.2 ± 0.2 s^− 1 for a random kinetic mechanism. PhzE can use NH₄⁺ as an alternative nucleophile, while Co^2 + and Mn^2 + are alternative divalent metals.     

Crystal structure of a Bombyx mori sigma-class glutathione transferase exhibiting prostaglandin E synthase activity

Background

Glutathione transferases (GSTs) are members of a major family of detoxification enzymes. Here, we report the crystal structure of a sigma-class GST of Bombyx mori, bmGSTS1, to gain insight into the mechanism catalysis.

Methods

The structure of bmGSTS1 and its complex with glutathione were determined at resolutions of 1.9 Å and 1.7 Å by synchrotron radiation and the molecular replacement method.

Results

The three-dimensional structure of bmGSTS1 shows that it exists as a dimer and is similar in structure to other GSTs with respect to its secondary and tertiary structures. Although striking similarities to the structure of prostaglandin D synthase were also detected, we were surprised to find that bmGSTS1 can convert prostaglandin H₂ into its E₂ form. Comparison of bmGSTS1 with its glutathione complex showed that bound glutathione was localized to the glutathione-binding site (G-site). Site-directed mutagenesis of bmGSTS1 mutants indicated that amino acid residues Tyr8, Leu14, Trp39, Lys43, Gln50, Met51, Gln63, and Ser64 in the G-site contribute to catalytic activity.

Conclusion

We determined the tertiary structure of bmGSTS1 exhibiting prostaglandin E synthase activity.

General significance

These results are, to our knowledge, the first report of a prostaglandin synthase activity in insects.     

Crystal Structures of Mutant Forms of the Yeast F1 ATPase Reveal Two Modes of Uncoupling

The mitochondrial ATP synthase couples the flow of protons with the phosphorylation of ADP. A class of mutations, the mitochondrial genome integrity (mgi) mutations, has been shown to uncouple this process in the yeast mitochondrial ATP synthase. Four mutant forms of the yeast F₁ ATPase with mgi mutations were crystallized; the structures were solved and analyzed. The analysis identifies two mechanisms of structural uncoupling: one in which the empty catalytic site is altered and in doing so, apparently disrupts substrate (phosphate) binding, and a second where the steric hindrance predicted between γLeu83 and β_DP residues, Leu-391 and Glu-395, located in Catch 2 region, is reduced allowing rotation of the γ-subunit with less impedance. Overall, the structures provide key insights into the critical interactions in the yeast ATP synthase involved in the coupling process.