FAD Binding Research Articles

A new F131V mutation in Chlamydomonas phytoene desaturase locates a cluster of norflurazon resistance mutations near the FAD-binding site in 3D protein models.

The green alga Chlamydomonas reinhardtii provides a tractable genetic model to study herbicide mode of action using forward genetics. The herbicide norflurazon inhibits phytoene desaturase, which is required for carotenoid synthesis. Locating amino acid substitutions in mutant phytoene desaturases conferring norflurazon resistance provides a genetic approach to map the herbicide binding site. We isolated a UV-induced mutant able to grow in very high concentrations of norflurazon (150 µM). The phytoene desaturase gene in the mutant strain contained the first resistance mutation to be localised to the dinucleotide-binding Rossmann-likedomain. A highly conserved phenylalanine amino acid at position 131 of the 564 amino acid precursor protein was changed to a valine in the mutant protein. F131, and two other amino acids whose substitution confers norflurazon resistance in homologous phytoene desaturase proteins, map to distant regions in the primary sequence of the C. reinhardtii protein (V472, L505) but in tertiary models these residues cluster together to a region close to the predicted FAD binding site. The mutant gene allowed direct 5 µM norflurazon based selection of transformants, which were tolerant to other bleaching herbicides including fluridone, flurtamone, and diflufenican but were more sensitive to beflubutamid than wild type cells. Norflurazon resistance and beflubutamid sensitivity allow either positive or negative selection against transformants expressing the mutant phytoene desaturase gene.

Interaction of Circadian Clock Proteins CRY1 and PER2 Is Modulated by Zinc Binding and Disulfide Bond Formation

Period (PER) proteins are essential components of the mammalian circadian clock. They form complexes with cryptochromes (CRY), which negatively regulate CLOCK/BMAL1-dependent transactivation of clock and clock-controlled genes. To define the roles of mammalian CRY/PER complexes in the circadian clock, we have determined the crystal structure of a complex comprising the photolyase homology region of mouse CRY1 (mCRY1) and a C-terminal mouse PER2 (mPER2) fragment. mPER2 winds around the helical mCRY1 domain covering the binding sites of FBXL3 and CLOCK/BMAL1, but not the FAD binding pocket. Our structure revealed an unexpected zinc ion in one interface, which stabilizes mCRY1-mPER2 interactions in vivo. We provide evidence that mCRY1/mPER2 complex formation is modulated by an interplay of zinc binding and mCRY1 disulfide bond formation, which may be influenced by the redox state of the cell. Our studies may allow for the development of circadian and metabolic modulators.

An alcohol oxidase of Phanerochaete chrysosporium with a distinct glycerol oxidase activity

An intracellular alcohol oxidase (AOX) was isolated from the white-rot basidiomycete Phanerochaete chrysosporium (Pch), grown on l-lactate induction medium, and purified to electrophoretic homogeneity. The dimeric protein consisted of two identical 75kDa subunits. The open reading frame of 1,956bp resulted in a monomer consisting of 651 amino acids. The enzyme showed a pI at 5.4, a pH optimum of 9, a temperature optimum at 50°C, possessed putative conserved domains of the GMC superfamily, a FAD binding domain, and showed up to 86% homology to alcohol oxidase sequences of Gloeophyllum trabeum and Coprinopsis cinerea. As was shown for the first time for an AOX from a basidiomycete, not only methanol, but also lower primary alcohols and glycerol were accepted as substrates. An assay based on aldehyde dehydrogenase confirmed d-glyceraldehyde as the product of the reaction. A bioprocess based on this enzyme could alleviate the problems associated with the huge side-stream of glycerol occurring during the manufacture of biodiesel, yielding the green oxidant hydrogen peroxide.

Structural and Functional Insights into the Catalytic Inactivity of the Major Fraction of Buffalo Milk Xanthine Oxidoreductase

BackgroundXanthine oxidoreductase (XOR) existing in two interconvertible forms, xanthine dehydrogenase (XDH) and xanthine oxidase (XO), catabolises xanthine to uric acid that is further broken down to antioxidative agent allantoin. XOR also produces free radicals serving as second messenger and microbicidal agent. Large variation in the XO activity has been observed among various species. Both hypo and hyper activity of XOR leads to pathophysiological conditions. Given the important nutritional role of buffalo milk in human health especially in south Asia, it is crucial to understand the functional properties of buffalo XOR and the underlying structural basis of variations in comparison to other species.Methods and FindingsBuffalo XO activity of 0.75 U/mg was almost half of cattle XO activity. Enzymatic efficiency (k cat/K m) of 0.11 sec−1 µM−1 of buffalo XO was 8–10 times smaller than that of cattle XO. Buffalo XOR also showed lower antibacterial activity than cattle XOR. A CD value (Δε430 nm) of 46,000 M−1 cm−1 suggested occupancy of 77.4% at Fe/S I centre. Buffalo XOR contained 0.31 molybdenum atom/subunit of which 48% existed in active sulfo form. The active form of XO in buffalo was only 16% in comparison to ∼30% in cattle. Sequencing revealed 97.4% similarity between buffalo and cattle XOR. FAD domain was least conserved, while metal binding domains (Fe/S and Molybdenum) were highly conserved. Homology modelling of buffalo XOR showed several variations occurring in clusters, especially close to FAD binding pocket which could affect NAD+ entry in the FAD centre. The difference in XO activity seems to be originating from cofactor deficiency, especially molybdenum.ConclusionA major fraction of buffalo milk XOR exists in a catalytically inactive form due to high content of demolybdo and desulfo forms. Lower Fe/S content and structural factors might be contributing to lower enzymatic efficiency of buffalo XOR in a minor way.

Structure of the bacterial type II NADH dehydrogenase: a monotopic membrane protein with an essential role in energy generation

Non-proton pumping type II NADH dehydrogenase (NDH-2) plays a central role in the respiratory metabolism of bacteria, and in the mitochondria of fungi, plants and protists. The lack of NDH-2 in mammalian mitochondria and its essentiality in important bacterial pathogens suggests these enzymes may represent a potential new drug target to combat microbial pathogens. Here, we report the first crystal structure of a bacterial NDH-2 enzyme at 2.5 Å resolution from Caldalkalibacillus thermarum. The NDH-2 structure reveals a homodimeric organization that has a unique dimer interface. NDH-2 is localized to the cytoplasmic membrane by two separated C-terminal membrane-anchoring regions that are essential for membrane localization and FAD binding, but not NDH-2 dimerization. Comparison of bacterial NDH-2 with the yeast NADH dehydrogenase (Ndi1) structure revealed non-overlapping binding sites for quinone and NADH in the bacterial enzyme. The bacterial NDH-2 structure establishes a framework for the structure-based design of small-molecule inhibitors.

FAD binding properties of a cytosolic version of Escherichia coli NADH dehydrogenase-2

Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a peripheral membrane-bound flavoprotein. By eliminating its C-terminal region, a water soluble truncated version was obtained in our laboratory. Overall conformation of the mutant version resembles the wild-type protein. Considering these data and the fact that the mutant was obtained as an apo-protein, the truncated version is an ideal model to study the interaction between the enzyme and its cofactor. Here, the FAD binding properties of this version were characterized using far-UV circular dichroism (CD), differential scanning calorimetry (DSC), limited proteolysis, and steady-state and dynamic fluorescence spectroscopy. CD spectra, thermal unfolding and DSC profiles did not reveal any major difference in secondary structure between apo- and holo-protein. In addition, digestion site accessibility and tertiary conformation were similar for both proteins, as seen by comparable chymotryptic cleavage patterns. FAD binding to the apo-protein produced a parallel increment of both FAD fluorescence quantum yield and steady-state emission anisotropy. On the other hand, addition of FAD quenched the intrinsic fluorescence emission of the truncated protein, indicating that the flavin cofactor should be closely located to the protein Trp residues. Analysis of the steady-state and dynamic fluorescence data confirms the formation of the holo-protein with a 1:1 binding stoichiometry and an association constant KA=7.0(±0.8)×104M−1. Taken together, the FAD–protein interaction is energetically favorable and the addition of FAD is not necessary to induce the enzyme folded state. For the first time, a detailed characterization of the flavin:protein interaction was performed among alternative NADH dehydrogenases.

Cloning, functional characterization, and expression profiles of NADPH-cytochrome P450 reductase gene from the Asiatic rice striped stem borer, Chilo suppressalis (Lepidoptera: Pyralidae)

NADPH-cytochrome P450 reductase (CPR) is one of the most important components of the cytochrome P450 enzyme system. It catalyzes electron transfer from NADPH to all known P450s, thus plays central roles not only in the metabolism of exogenous xenobiotics but also in the regulation of endogenous hormones in insects. In this study, a full-length cDNA encoding of a CPR (named CsCPR) was isolated from the Asiatic rice striped stem borer, Chilo suppressalis, by using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The cDNA contains a 2061 bp open reading frame, which encodes an enzyme of 686 amino acid residues, with a calculated molecular mass of 77.6 kDa. The deduced peptide has hallmarks of typical CPR, including an N-terminal membrane anchor and the FMN, FAD and NADPH binding domains. The N-terminal-truncated protein fused with a 6 × His·tag was heterologously expressed in Escherichia coli Rosetta (DE3) cells and purified, specific activity and the Km values of the recombinant enzyme were determined. Tissue- and developmental stage-dependent expression of CsCPR mRNA was investigated by real-time quantitative PCR. The CsCPR mRNA was noticeably expressed in the digestive, metabolic, and olfactory organs of the larvae and adults of C. suppressalis. Our initial results would provide valuable information for further study on the interactions between CPR and cytochrome P450 enzyme systems.

MD and QM/MM study on catalytic mechanism of a FAD-dependent enzyme ORF36: For nitro sugar biosynthesis

The catalytic mechanism of a FAD-dependent nitrososynthase (ORF36) was studied with molecular dynamics (MD) and quantum mechanical/molecular mechanical (QM/MM) methods. Residues Leu160 and Phe374 play an important role during the FAD binding with ORF36. Similar phenylalanine/leucine pair was found in the other two enzymes of this family. For the second oxidation step of ORF36 toward thymidine diphosphate-l-epi-vancosamine, three elementary catalytic steps were found: a hydroxylation step, a hydrogen back-transfer step and a hydroxyl group elimination step. The hydroxylation step is found to be the rate-determining step with an energy barrier of 26.3kcal/mol under the B3LYP/cc-pVTZ//CHARMM22 level. Two possible pathways for the second oxidation step are carefully investigated. Our simulations indicate that an oxygen atom from the coenzyme FADHOOH is inserted into the product. In addition, the electrostatic influence of 17 individual residues and five neighboring water molecules on the rate-determining step was estimated. The results indicate that groups Gly132/Ala133/Leu134, Met375/Gln376 and a water fence play a key role in facilitating the rate-determining step. On the other hand, residues Leu160, Val161 and Ser162 are found to be critical to suppress the rate-determining step. Our results lead to further understanding of the detailed catalytic pathways for nitro sugar biosynthesis.

Chloroquine Binding Reveals Flavin Redox Switch Function of Quinone Reductase 2

Quinone reductase 2 (NQO2) is an FAD-linked enzyme and the only known human target of two antimalarial drugs, primaquine (PQ) and chloroquine (CQ). The structural differences between oxidized and reduced NQO2 and the structural basis for inhibition by PQ and CQ were investigated by x-ray crystallography. Structures of oxidized NQO2 in complex with PQ and CQ were solved at 1.4 Å resolution. CQ binds preferentially to reduced NQO2, and upon reduction of NQO2-CQ crystals, the space group changed from P2(1)2(1)2(1) to P2(1), with 1-Å decreases in all three unit cell dimensions. The change in crystal packing originated in the negative charge and 4-5º bend in the reduced isoalloxazine ring of FAD, which resulted in a new mode of CQ binding and closure of a flexible loop (Phe(126)-Leu(136)) over the active site. This first structure of a reduced quinone reductase shows that reduction of the FAD cofactor and binding of a specific inhibitor lead to global changes in NQO2 structure and is consistent with a functional role for NQO2 as a flavin redox switch.

Evolution study of the Baeyer–Villiger monooxygenases enzyme family: Functional importance of the highly conserved residues

Baeyer-Villiger monooxygenases (BVMOs) catalyze the transformation of linear and cyclic ketones into their corresponding esters and lactones by introducing an oxygen atom into a C-C bond. This bioreaction has numerous advantages compared to its chemical version; it does not induce the use of potentially harmful reagents (i.e., green chemistry) and displays significant better enantio- and regio-selectivity. New potential BVMOs were searched using sequence homology for type I BVMO proteins. 116 new sequences were identified as new putative BVMOs respecting the defined selection criteria. Multiple sequence alignments were carried out on the selected sequences to study the conservation of structurally and/or functionally important amino acids during evolution. Type I BVMO signature motif was found to be conserved in 94.8% of the sequences. We noticed also the highly conserved - but previously unnoticed - Threonine 167 (93.1%), located in the signature motif; this position could be added in the pattern used to characterize specific Type I enzymes. Amino acids at the vicinity of the FAD and NADPH cofactors were found also to be highly conserved and the details of the interactions were emphasized. Interestingly, residues at the enzyme binding site were found less conserved in terms of sequence evolution, leading sometimes to some important amino acid changes. These behaviors could explain the enzyme selectivity and specificity for different ligands.

Flavinylation and assembly of succinate dehydrogenase are dependent on the C-terminal tail of the flavoprotein subunit.

Succinate dehydrogenase (SDH) requires a covalent addition of FAD for catalytic function. Mutational analyses of Sdh1 implicate C-terminal region Arg residues involvement in covalent flavinylation and SDH assembly. SDH assembly is dependent on FAD binding to Sdh1 but not covalent binding. These results document the basis for the SDH deficiency and pathology seen with mutations in human Sdh1. The enzymatic function of succinate dehydrogenase (SDH) is dependent on covalent attachment of FAD on the ~70-kDa flavoprotein subunit Sdh1. We show presently that flavinylation of the Sdh1 subunit of succinate dehydrogenase is dependent on a set of two spatially close C-terminal arginine residues that are distant from the FAD binding site. Mutation of Arg(582) in yeast Sdh1 precludes flavinylation as well as assembly of the tetrameric enzyme complex. Mutation of Arg(638) compromises SDH function only when present in combination with a Cys(630) substitution. Mutations of either Arg(582) or Arg(638)/Cys(630) do not markedly destabilize the Sdh1 polypeptide; however, the steady-state level of Sdh5 is markedly attenuated in the Sdh1 mutant cells. With each mutant Sdh1, second-site Sdh1 suppressor mutations were recovered in Sdh1 permitting flavinylation, stabilization of Sdh5 and SDH tetramer assembly. SDH assembly appears to require FAD binding but not necessarily covalent FAD attachment. The Arg residues may be important not only for Sdh5 association but also in the recruitment and/or guidance of FAD and or succinate to the substrate site for the flavinylation reaction. The impaired assembly of SDH with the C-terminal Sdh1 mutants suggests that FAD binding is important to stabilize the Sdh1 conformation enabling association with Sdh2 and the membrane anchor subunits.

Crystal Structure of a Flavin-dependent Thymidylate Synthase from Helicobacter pylori strain 26695

ThyX, a flavin-dependent thymidylate synthase that is involved in the synthesis of dTMP from dUMP, is a promising target for the development of novel antibacterial drugs that aimed at blocking the biosynthesis of dTMP, one of the building blocks of DNA. This enzyme has been recently identified in some dsDNA viruses and pathogenic bacteria, including the gastric pathogen Helicobacter pylori. It shares neither sequence nor structural homology with the classical ThyA in humans and other organisms. Further more, ThyX and ThyA are the only source of dTMP in these organisms and other pathways cannot substitute for their function. Thus, ThyX-specific inhibitors could be effective antibacterial reagents while having no impact on human cells. Here we report the crystal structure of ThyX from Helicobacter pylori strain 26695 in complex with co-factor FAD and substrate dUMP at 2.5 A resolution, which consists of a 1.5 tetramer of ThyX with a total of 1248 residues, six FAD and six dUMP molecules in an asymmetric unit. The structure revealed the key residues that are involved in co-factor FAD and substrate dUMP binding, site-directed mutagenesis were performed to analysis the importance of these residues on ThyX activity by genetic complementation and FAD binding assay.

The thioredoxin reductase-encoding gene ActrxR1 is involved in the cephalosporin C production of Acremonium chrysogenum in methionine-supplemented medium

The thioredoxin system including thioredoxin and thioredoxin reductase (TrxR) is used for oxidative stress defenses in fungi. Based on the genomic sequence, a thioredoxin reductase-encoding gene (ActrxR1) was isolated from Acremonium chrysogenum CGMCC3.3795. Like other TrxRs, AcTrxR1 contains FAD binding domain, Redox domain, and NADPH binding domain. Disruption of ActrxR1 in A. chrysogenum led to the formation of smaller colonies and hyphal swelling in Tryptic soy agar (TSA). In chemically defined medium, the spore germination of ActrxR1 disruption mutant was strongly inhibited, which was recovered by the addition of DL-methionine. The disruption mutant grew slowly on TSA compared with the wild-type strain, but it did not show to be more sensitive to exogenous hydrogen peroxide or menadione. In defined medium of fermentation supplemented with DL-methionine, the ActrxR1 disruption mutant grew normally, and its cephalosporin C production increased by about onefold compared with the wild type (73 μg/ml for wild-type strain and 136 μg/ml for the mutant at 5 days of fermentation). Real-time polymerase chain reaction (RT-PCR) showed that the transcriptional levels of pcbC, cefEF, and cefG were obviously enhanced in the ActrxR1 mutant at the early stage of fermentation. These results indicate that ActrxR1 is required for the normal growth of A. chrysogenum and related with cephalosporin C production in methionine-supplemented medium.

An Unconventional Hexacoordinated Flavohemoglobin from Mycobacterium tuberculosis

Being an obligate aerobe, Mycobacterium tuberculosis faces a number of energetic challenges when it encounters hypoxia and environmental stress during intracellular infection. Consequently, it has evolved innovative strategies to cope with these unfavorable conditions. Here, we report a novel flavohemoglobin (MtbFHb) from M. tuberculosis that exhibits unique features within its heme and reductase domains distinct from conventional FHbs, including the absence of the characteristic hydrogen bonding interactions within the proximal heme pocket and mutations in the FAD and NADH binding regions of the reductase domain. In contrast to conventional FHbs, it has a hexacoordinate low-spin heme with a proximal histidine ligand lacking imidazolate character and a distal heme pocket with a relatively low electrostatic potential. Additionally, MtbFHb carries a new FAD binding site in its reductase domain similar to that of D-lactate dehydrogenase (D-LDH). When overexpressed in Escherichia coli or Mycobacterium smegmatis, MtbFHb remained associated with the cell membrane and exhibited D-lactate:phenazine methosulfate reductase activity and oxidized D-lactate into pyruvate by converting the heme iron from Fe(3+) to Fe(2+) in a FAD-dependent manner, indicating electron transfer from D-lactate to the heme via FAD cofactor. Under oxidative stress, MtbFHb-expressing cells exhibited growth advantage with reduced levels of lipid peroxidation. Given the fact that D-lactate is a byproduct of lipid peroxidation and that M. tuberculosis lacks the gene encoding D-LDH, we propose that the novel D-lactate metabolizing activity of MtbFHb uniquely equips M. tuberculosis to balance the stress level by protecting the cell membrane from oxidative damage via cycling between the Fe(3+)/Fe(2+) redox states.

Fluorescence detection of obligatory conformations in catalysis and control of NOS

Nitric Oxide synthases produce NO as a signal and as a cytotoxin in immune response. In the tethered shuttle model for NOS reductase kinetics, the FMN domain moves between NADPH dehydrogenase and oxygenase catalytic sites. FMN fluorescence is sensitive to NOS conformation; eNOS and nNOS holoenzymes have states with lifetimes of ~4.3ns, 1 ns, and 90 ps. Two domain oxyFMN constructs (truncated before the FAD binding domain) and holoenzyme share open conformations with lifetimes of ~4.3 ns. The holoenzyme majority state has a lifetime of ~90 ps due to FAD/FMN proximity as in crystal structures (Wang et al, Garcin et al). In oxyFMN ~25% of FMN has a lifetime of 0.9 ns, which we attribute to quenching by heme. Calmodulin activation of NOS releases the FMN binding domain from the ‘dehydrogenase’ complex, increasing the populations of the long lifetime states. Calmodulin binding shortens the average lifetime of oxyFMN constructs, because CaM promotes association of the FMN binding domain with the oxygenase domain, and favors the 1 ns state over the 4.3 ns state. Similar effects help explain modulation of NOS activity by covalent modification and protein‐protein interactions, e.g., in the HSP90‐eNOS complex.

Unveiling the Post-PKS Redox Tailoring Steps in Biosynthesis of the Type II Polyketide Antitumor Antibiotic Xantholipin

Xantholipin from Streptomyces flavogriseus isa curved hexacyclic aromatic polyketide antitumor antibiotic. The entire 52 kb xantholipin (xan) biosynthetic gene cluster was sequenced, and bioinformatic analysis revealed open reading frames encoding type II polyketide synthases, regulators, and polyketide tailoring enzymes. Individual in-frame mutagenesis of five tailoring enzymes lead to the production of nine xantholipin analogs, revealing that the xanthone scaffold formation was catalyzed by the FAD binding monooxygenase XanO4, the δ-lactam formation by the asparagine synthetase homolog XanA, the methylenedioxy bridge generation by the P450 monooxygenase XanO2 and the hydroxylation of the carbon backbone by the FAD binding monooxygenase XanO5. These findings may also apply to other polycyclic xanthone antibiotics, and they form the basis for genetic engineering of the xantholipin and similar biosynthetic gene clusters for the generation of compounds with improved antitumor activities.

RNA Interference of NADPH-Cytochrome P450 Reductase Results in Reduced Insecticide Resistance in the Bed Bug, Cimex lectularius

BackgroundNADPH-cytochrome P450 reductase (CPR) plays a central role in cytochrome P450 action. The genes coding for P450s are not yet fully identified in the bed bug, Cimex lectularius. Hence, we decided to clone cDNA and knockdown the expression of the gene coding for CPR which is suggested to be required for the function of all P450s to determine whether or not P450s are involved in resistance of bed bugs to insecticides.Methodology/Principal FindingsThe full length Cimex lectularius CPR (ClCPR) cDNA was isolated from a deltamethrin resistant bed bug population (CIN-1) using a combined PCR strategy. Bioinformatics and in silico modeling were employed to identify three conserved binding domains (FMN, FAD, NADP), a FAD binding motif, and the catalytic residues. The critical amino acids involved in FMN, FAD, NADP binding and their putative functions were also analyzed. No signal peptide but a membrane anchor domain with 21 amino acids which facilitates the localization of ClCPR on the endoplasmic reticulum was identified in ClCPR protein. Phylogenetic analysis showed that ClCPR is closer to the CPR from the body louse, Pediculus humanus corporis than to the CPRs from the other insect species studied. The ClCPR gene was ubiquitously expressed in all tissues tested but showed an increase in expression as immature stages develop into adults. We exploited the traumatic insemination mechanism of bed bugs to inject dsRNA and successfully knockdown the expression of the gene coding for ClCPR. Suppression of the ClCPR expression increased susceptibility to deltamethrin in resistant populations but not in the susceptible population of bed bugs.Conclusions/SignificanceThese data suggest that P450-mediated metabolic detoxification may serve as one of the resistance mechanisms in bed bugs.

Targeting and Maturation of Erv1/ALR in the Mitochondrial Intermembrane Space

The interaction of Mia40 with Erv1/ALR is central to the oxidative protein folding in the intermembrane space of mitochondria (IMS) as Erv1/ALR oxidizes reduced Mia40 to restore its functional state. Here we address the role of Mia40 in the import and maturation of Erv1/ALR. The C-terminal FAD-binding domain of Erv1/ALR has an essential role in the import process by creating a transient intermolecular disulfide bond with Mia40. The action of Mia40 is selective for the formation of both intra and intersubunit structural disulfide bonds of Erv1/ALR, but the complete maturation process requires additional binding of FAD. Both of these events must follow a specific sequential order to allow Erv1/ALR to reach the fully functional state, illustrating a new paradigm for protein maturation in the IMS.

Cloning and sequence analysis on cDNA of squalene epoxidase gene in Eleutherococcus senticosus

To clone and sequence the cDNA of squalene epoxidase gene in Eleutherococcus senticosus. Total RNA of E. senticosus was extracted by the improved isothiocyanate method and reverse transcripted into cDNA. The primers were designed depending on the reported SE cDNA sequences of Panax ginseng. The SE cDNAs in E. senticosus was amplified using RT-PCR strategy. Sequencing results showed two different cDNA fragments (SE1, SE2) with 1665, 1629 bp each ORF which encoded 554,542 amino acids, respectively. The identities of nucleotides and amino acids between SE1, SE2 were 91.49%, 92.55%. SE1, SE2 had the highest amino acids similarity to the SE1 of P. notoginseng, 93.45%, 94.87% respectively. SE1, SE2 both had a FAD binding domain. The deduced speculated amino acids of SE1, SE2 each had 2,4 membrane-spanning helices. The two SE sequences in E. senticosus were firstly separated and reported, which has made foundation for E. senticosus secondary metabolite engineering researches.

Investigation of trypanothione reductase inhibitory activity by 1,3,4-thiadiazolium-2-aminide derivatives and molecular docking studies

The biological activities of a series of mesoionic 1,3,4-thiadiazolium-2-aminide derivatives have been studied. The most active compounds (MI-HH; MI-3-OCH3; MI-4-OCH3 and MI-4-NO2) were evaluated to determine their effect on trypanothione reductase (TryR) activity in Leishmania sp. and Trypanosoma cruzi. Among the assayed compounds, only MI-4-NO2 showed enzyme inhibition effect on extracts from different cultures of parasites, which was confirmed using the recombinant enzyme from T. cruzi (TcTryR) and Leishmania infantum (LiTryR). The enzyme kinetics determined with LiTryR demonstrated a non-competitive inhibition profile of MI-4-NO2. A molecular docking study showed that the mesoionic compounds could effectively dock into the substrate binding site together with the substrate molecule. The mesoionic compounds were also effective ligands of the NADPH and FAD binding sites and the NADPH binding site was predicted as the best of all three binding sites. Based on the theoretical results, an explanation at the molecular level is proposed for the MI-4-NO2 enzyme inhibition effect. Given TryR as a molecular target, it is important to continue the study of mesoionic compounds as part of a drug discovery campaign against Leishmaniasis or Chagas’ disease.