Chemical structures of polyketides and alkaloids isolated from a culture of fungus Curvularia moringae.

Seven new polyketides [moringols I-VII (1-7)], a new alkaloid [moringamine I (8)], and seven known compounds (9-15) were isolated from the fungus Curvularia moringae JKYM-KR4. The planar chemical structures and relative configurations of the new compounds were elucidated by high-resolution mass spectrometry, 1D and 2D NMR spectroscopy, and DP4+ analysis using the calculated 13C NMR chemical shifts. For moringols I and II (1 and 2), the planar chemical structures and relative configurations were confirmed using X-ray crystallography. The absolute configurations of 1-6 and 8 were determined by ECD calculations. Among the isolated compounds, terpestacin (14) moderately inhibited the proliferation of HT-29 cells.

Substrate preference, RNA binding and active site versatility of Stenotrophomonas maltophilia nuclease SmNuc1, explained by a structural study.

Nucleases of the S1/P1 family have important applications in biotechnology and molecular biology. We have performed structural analyses of SmNuc1 nuclease from Stenotrophomonas maltophilia, including RNA cleavage product binding and mutagenesis in a newly discovered flexible Arg74-motif, involved in substrate binding and product release and likely contributing to the high catalytic rate. The Arg74Gln mutation shifts substrate preference towards RNA. Purine nucleotide binding differs compared to pyrimidines, confirming the plasticity of the active site. The enzyme-product interactions indicate a gradual, stepwise product release. The activity of SmNuc1 towards c-di-GMP in crystal resulted in a distinguished complex with the emerging product 5'-GMP. This enzyme from an opportunistic pathogen relies on specific architecture enabling high performance under broad conditions, attractive for biotechnologies.

Crystallization and crystallographic studies of human serine protease inhibitor (serpin) B9.

Serine protease inhibitor B9 (serpin B9, also known as protease inhibitor 9 or PI9) plays a critical role in regulating the immune response by specifically inhibiting granzyme B, a serine protease found in cytotoxic T lymphocytes and natural killer cells. Despite its potential as an anticancer drug target, the structural details of serpin B9 have remained elusive until now. In this study, a cleaved form of recombinant human serpin B9 was successfully prepared and crystallized. The crystals belonged to space group P212121, with unit-cell parameters a = 68.51, b = 82.32, c = 101.17 Å, and an X-ray diffraction data set was collected at 1.9 Šresolution. The structure shows that serpin B9 adopts a relaxed conformation, with its cleaved reactive-centre loop inserted into the central ß-sheet. Unlike other serpins, serpin B9 shows significant structural deviations around helix D, with a larger surface cavity, which could serve as a promising target for small-molecule inhibitors.

Elucidation of the stereochemical mechanism of cystathionine γ-lyase reveals how substrate specificity constrains catalysis.

Pyridoxal phosphate (PLP)-dependent enzymes play essential roles in metabolism and have found applications for organic synthesis and as enzyme therapeutics. The vinylglycine ketimine (VGK) subfamily hosts a growing set of enzymes that play diverse roles in primary and secondary metabolism. However, the molecular determinates of substrate specificity and the complex acid-base chemistry that enables VGK catalysis remain enigmatic. We use a recently discovered amino acid γ-lyase as a model system to probe catalysis in this enzyme family. We discovered that two stereochemically distinct proton transfer pathways occur. Combined kinetic and spectroscopic analysis revealed that progression through the catalytic cycle is correlated with the presence of an H-bond donor after Cγ of an amino acid substrate, suggesting substrate binding is kinetically coupled to a conformational change. High-resolution X-ray crystallography shows that cystathionine-γ-lyases generate an s-trans intermediate and that this geometry is likely conserved throughout the VGK family. An H-bond acceptor in the active site templates substrate binding but does so by pre-organizing substrates away from catalytically productive orientations. Mutagenesis eliminates this pre-organization, such that there is a relaxation of the substrate specificity, but an increase in k cat for diverse substrates. We exploit this information to perform preparative scale α,ß,ß-tri-deuteration of polar amino acids. Together, these data untangle a complex mode of substrate specificity and provide a foundation for the future study and applications of VGK enzymes.

Structural basis for the ligand promiscuity of the hydroxamate siderophore binding protein FtsB from Streptococcus pyogenes.

Pathogenic bacteria must secure the uptake of nutritional metals such as iron for their growth, making their import systems attractive targets for the development of new antimicrobial modalities. In the pathogenic bacterium Streptococcus pyogenes, the iron uptake system FtsABCD transports iron encapsulated by siderophores of the hydroxamate class. However, the inability of S. pyogenes to produce these metabolites makes the biological and clinical relevance of this route unresolved. Herein, we demonstrated that the periplasmic binding protein FtsB recognizes not only the hydroxamate siderophore ferrichrome, as previously documented, but also ferrioxamine E (FOE), ferrioxamine B (FOB), and bisucaberin (BIS), each of them with high affinity (nM level). Up to seven aromatic residues in the binding pocket accommodate the variable backbones of the different siderophores through CH-π interactions, explaining ligand promiscuity. Collectively, our observations revealed how S. pyogenes exploits the diverse xenosiderophores produced by other microorganisms as iron sources to secure this precious nutrient.

Tyramine Derivatives as Versatile Pharmacophores With Potent Biological Properties: Sex Hormone-Binding Globulin Inhibition, Colon Cancer Antimigration, and Antimicrobial Activity.

Guided by the idea that the presence of a heterocyclic aromatic core and tyramine moiety, under the umbrella of a single molecular scaffold could bring interesting biological properties, herein we present synthesis, characterization, with two crystal structures reported, and biological evaluation of some tyramine derivates. Cytotoxic and antimigratory potential was addressed by using a colorectal cancer cell line as a model system. Although possessing no cytotoxic effects, two compounds have shown strong antimigratory potential in low doses, with no effect on healthy MRC-5 cells. Evaluation of their antimicrobial activities suggested prominent antimicrobial activity, where Compound 4 outperformed streptomycin against Escherichia coli and Proteus mirabilis. Hormone-dependent types of cancer, such as prostate, ovary, and breast, are highly dependent on human sex hormone-binding globulin (SHBG) blood levels. A molecular docking study has shown that 1 has high affinity to bind and therefore compete with natural steroids for the SHBG steroid-binding site. DNA-binding study have shown that 4 interacts with CT-DNA in a groove-binding mode. In silico ADME/T study revealed that all compounds have suitable physicochemical properties for oral bioavailability and druglikeness, while toxicity tests for 1, 4, and 6 suggested potential for mutagenicity (4, 6), hepatotoxicity (6), and skin sensation (1).

Hirshfeld Atom Refinement (HAR) and Complementary Bonding Analysis of Lithium m-Terphenylhydridoborates Containing B-H···Li Linkages.

The synthesis and characterization of the lithium m-terphenylhydridoborates [Li(2,6-Mes2C6H3)BH3]2 (1), Li(2,6-Mes2C6H3)2BH2 (2) and Li(OEt2)(2,6-Mes2C6H3)2BH2 (3) are reported. Hirshfeld Atom Refinement (HAR) of the experimentally obtained molecular structures by single-crystal X-ray crystallography allowed the determination of the exact positions of the hydrogen atoms. The bond situations of the various B-H···Li linkages were investigated by a complementary bonding analysis using various methods including atoms in molecules (AIM), electron localizability indicator (ELI-D), non-covalent interaction (NCI) index and the compliance matrix.

Antimicrobial Activity of Anionic Bis(N-Heterocyclic Carbene) Silver Complexes.

The antimicrobial properties of a series of anionic bis(carbene) silver complexes Na3[Ag(NHCR)2] were investigated (2a-2g and 2c', where NHCR is a 2,2'-(imidazol-2-ylidene)dicarboxylate-type N-heterocyclic carbene). The complexes were synthesized by the interaction of imidazolium dicarboxylate compounds with silver oxide in the presence of aqueous sodium hydroxide. Complexes 2f,g were characterized analytically and spectroscopically, and the ligand precursor 1f and complexes 2c and 2g were structurally identified by X-ray diffraction methods. The anions of 2c and 2g, [Ag(NHCR)2]3-, showed a typical linear disposition of Ccarbene-Ag-Ccarbene atoms and an uncommonly eclipsed conformation of carbene ligands. The antimicrobial properties of complexes 2a-g, which contains chiral (2b-2e and 2c') and non-chiral derivatives (2a,f,g), were evaluated against Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa, and a Gram-positive bacterium, Staphylococcus aureus. From the observed values of the minimal inhibitory concentration and minimal bactericidal concentration, complexes 2a and 2b showed the best antimicrobial activity against all strains. An interesting chirality-antimicrobial relationship was found, and eutomer 2c' showed better activity than its enantiomer 2c against the three bacteria. Furthermore, these complexes were investigated experimentally and theoretically by 109Ag nuclear magnetic resonance, and the electronic and steric characteristics of the dianionic carbene ligands were also examined.

Creation of Metal-Complex-Integrated Tensegrity Triangle DNA Crystals.

Structural DNA nanotechnology is an emerging field and is expected to be used for various applications in materials science. In this study, we designed a DNA tensegrity triangle to accommodate the bipyridine complexes with metal ions (Ni2+ and Fe2+) at the center of the space within the triangle. A metal-bipyridine-incorporated DNA tensegrity triangle was crystalized, and the presence of metals within it was confirmed through X-ray crystal structure analysis. A signal of the anomalous dispersion effect derived from metal was observed in the center of the DNA triangle.

Interaction of Tri-Cyclic Nucleobase Analogs with Enzymes of Purine Metabolism: Xanthine Oxidase and Purine Nucleoside Phosphorylase.

Fluorescent markers play important roles in spectroscopic and microscopic research techniques and are broadly used in basic and applied sciences. We have obtained markers with fluorescent properties, two etheno derivatives of 2-aminopurine, as follows: 1,N2-etheno-2-aminopurine (1,N2-ε2APu, I) and N2,3-etheno-2-aminopurine (N2,3-ε2APu, II). In the present paper, we investigate their interaction with two key enzymes of purine metabolism, purine nucleoside phosphorylase (PNP), and xanthine oxidase (XO), using diffraction of X-rays on protein crystals, isothermal titration calorimetry, and fluorescence spectroscopy. Crystals were obtained and structures were solved for WT PNP and D204N-PNP mutant in a complex with N2,3-ε2APu (II). In the case of WT PNP-1,N2-ε2APu (I) complex, the electron density corresponding to the ligand could not be identified in the active site. Small electron density bobbles may indicate that the ligand binds to the active site of a small number of molecules. On the basis of spectroscopic studies in solution, we found that, in contrast to PNP, 1,N2-ε2APu (I) is the ligand with better affinity to XO. Enzymatic oxidation of (I) leads to a marked increase in fluorescence near 400 nm. Hence, we have developed a new method to determine XO activity in biological material, particularly suitable for milk analysis.

Open and closed structures of L-arginine oxidase by cryo-electron microscopy and X-ray crystallography.

L-arginine oxidase (AROD, EC 1.4.3.25) is an oxidoreductase that catalyzes the deamination of L-arginine, with flavin adenine dinucleotide (FAD) as a cofactor. Recently identified AROD from Pseudomonas sp. TPU 7192 (PT-AROD) demonstrates high selectivity for L-arginine. This enzyme is useful for accurate assays of L-arginine in biological samples. The structural characteristics of the FAD-dependent AROD, however, remain unknown. Here, we report the structure of PT-AROD at a resolution of 2.3 Å by cryo-electron microscopy. PT-AROD adopts an octameric structure with D4 symmetry, which is consistent with its molecular weight in solution, estimated by mass photometry. Comparative analysis of this structure with that determined using X-ray crystallography reveals open and closed forms of the lid-like loop at the entrance to the substrate pocket. Furthermore, mutation of Glu493, located at the substrate binding site, diminishes substrate selectivity, suggesting that this residue contributes significantly to the high selectivity of PT-AROD.

dUTP pyrophosphatases from hyperthermophilic eubacterium and archaeon: Structural and functional examinations on the suitability for PCR application.

Deoxyuridine triphosphate pyrophosphatase (DUT) suppresses incorporation of uracil into genomic DNA during replication. Thermostable DUTs from hyperthermophilic archaea such as Thermococcus pacificus enhance PCR amplification by preventing misincorporation of dUTP generated by spontaneous deamination of dCTP. However, it is necessary to elucidate whether DUTs do not cause dNTP imbalances during PCR by unwanted side activity. Moreover, it has been unknown what structural features define the thermostability of those DUTs. Here, DUT from a hyperthermophilic eubacterium, Aquifex aeolicus (Aa-DUT), was characterized together with those from T. pacificus (Tp-DUT). Aa-DUT was as thermostable as Tp-DUT up to at least 95°C. The crystal structures of the two thermostable enzymes were determined, which revealed that the structures of Aa-DUT and Tp-DUT resembled those of monofunctional and bifunctional DUTs, respectively. Generally, bifunctional DUTs harbor the dCTP deaminase activity in addition to the DUT activity. However, not only Aa-DUT but also Tp-DUT showed poor activity towards dCTP, indicating both enzymes are monofunctional. We further examined eight types of parameters related to thermostability of protein structure and found that the thermostability of Aa-DUT and Tp-DUT might be accomplished by increased numbers of ion pairs on the protein surface. Finally, we verified that Aa-DUT promoted PCR amplification with Pfu DNA polymerase to the same extent as Tp-DUT. Collectively, we conclude that both DUTs from hyperthermophiles maintain the enzymatic activity at high temperatures without consuming dCTP due to the lack of the deaminate activity.

Mechanism of phospho-Ubls' specificity and conformational changes that regulate Parkin activity.

PINK1 and Parkin mutations lead to the early onset of Parkinson's disease. PINK1-mediated phosphorylation of ubiquitin (Ub), ubiquitin-like protein (NEDD8), and ubiquitin-like (Ubl) domain of Parkin activate autoinhibited Parkin E3 ligase. The mechanism of various phospho-Ubls' specificity and conformational changes leading to Parkin activation remain elusive. Herein, we show that compared to Ub, NEDD8 is a more robust binder and activator of Parkin. Structures and biophysical/biochemical data reveal specific recognition and underlying mechanisms of pUb/pNEDD8 and pUbl domain binding to the RING1 and RING0 domains, respectively. Also, pUb/pNEDD8 binding in the RING1 pocket promotes allosteric conformational changes in Parkin's catalytic domain (RING2), leading to Parkin activation. Furthermore, Parkinson's disease mutation K211N in the RING0 domain was believed to perturb Parkin activation due to loss of pUb binding. However, our data reveal allosteric conformational changes due to N211 that lock RING2 with RING0 to inhibit Parkin activity without disrupting pNEDD8/pUb binding.

Structural studies of ß-glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharolyticus.

ß-Glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharolyticus (Bgl1) has been denoted as having an attractive catalytic profile for various industrial applications. Bgl1 catalyses the final step of in the decomposition of cellulose, an unbranched glucose polymer that has attracted the attention of researchers in recent years as it is the most abundant renewable source of reduced carbon in the biosphere. With the aim of enhancing the thermostability of Bgl1 for a broad spectrum of biotechnological processes, it has been subjected to structural studies. Crystal structures of Bgl1 and its complex with glucose were determined at 1.47 and 1.95 Šresolution, respectively. Bgl1 is a member of glycosyl hydrolase family 1 (GH1 superfamily, EC 3.2.1.21) and the results showed that the 3D structure of Bgl1 follows the overall architecture of the GH1 family, with a classical (ß/α)8 TIM-barrel fold. Comparisons of Bgl1 with sequence or structural homologues of ß-glucosidase reveal quite similar structures but also unique structural features in Bgl1 with plausible functional roles.

Robust and automatic beamstop shadow outlier rejection: combining crystallographic statistics with modern clustering under a semi-supervised learning strategy.

During the automatic processing of crystallographic diffraction experiments, beamstop shadows are often unaccounted for or only partially masked. As a result of this, outlier reflection intensities are integrated, which is a known issue. Traditional statistical diagnostics have only limited effectiveness in identifying these outliers, here termed Not-Excluded-unMasked-Outliers (NEMOs). The diagnostic tool AUSPEX allows visual inspection of NEMOs, where they form a typical pattern: clusters at the low-resolution end of the AUSPEX plots of intensities or amplitudes versus resolution. To automate NEMO detection, a new algorithm was developed by combining data statistics with a density-based clustering method. This approach demonstrates a promising performance in detecting NEMOs in merged data sets without disrupting existing data-reduction pipelines. Re-refinement results indicate that excluding the identified NEMOs can effectively enhance the quality of subsequent structure-determination steps. This method offers a prospective automated means to assess the efficacy of a beamstop mask, as well as highlighting the potential of modern pattern-recognition techniques for automating outlier exclusion during data processing, facilitating future adaptation to evolving experimental strategies.

Random mutagenesis and disulfide bond formation improved thermostability in microbial transglutaminase.

Microbial transglutaminase (MTG) from Streptomyces mobaraensis is widely used in the food and pharmaceutical industries for cross-linking and post-translational modification of proteins. It is believed that its industrial applications could be further broadened by improving its thermostability. In our previous study, we showed that the introduction of structure-based disulfide bonds improved the thermostability of MTG, and we succeeded in obtaining a thermostable mutant, D3C/G283C, with a T50 (incubation temperature at which 50% of the initial activity remains) 9 °C higher than that of wild-type MTG. In this study, we performed random mutations using D3C/G283C as a template and found several amino acid substitutions that contributed to the improvement of thermostability, and investigated a thermostable mutant (D3C/S101P/G157S/G250R/G283C) with three amino acid mutations in addition to the disulfide bond. The T50 of this mutant was 10 °C higher than that of the wild type, the optimal temperature for enzymatic reaction was increased to 65 °C compared to 50 °C for the wild type, and the catalytic efficiency (kcat/Km) at 37.0 °C was increased from 3.3 × 102 M-1 s-1 for the wild type to 5.9 × 102 M-1 s-1. X-ray crystallography of the D3C/G283C MTG showed no major structural differences against wild-type MTG. Structural differences were found that may contribute to thermostabilization and improve catalytic efficiency. KEY POINTS: • Improved heat resistance is essential to broaden the application of MTG. • The MTG mutant D3C/S101P/G157S/G250R/G283C showed improved thermostability. • X-ray crystallography of the disulfide bridge mutant D3C/G283C MTG was elucidated.

Structural characterization of green fluorescent protein in the I-state.

Green fluorescent protein (GFP) is widely utilized as a fluorescent tag in biochemical fields. Whereas the intermediate (I) state has been proposed in the photoreaction cycle in addition to the A and B states, until now the structure of I has only been estimated by computational studies. In this paper, we report the crystal structures of the I stabilizing variants of GFP at high resolutions where respective atoms can be observed separately. Comparison with the structures in the other states highlights the structural feature of the I state. The side chain of one of the substituted residues, Val203, adopts the gauche- conformation observed for Thr203 in the A state, which is different from the B state. On the other hand, His148 interacts with the chromophore by ordinary hydrogen bonding with a distance of 2.85 Å, while the weaker interaction by longer distances is observed in the A state. Therefore, it was indicated that it is possible to distinguish three states A, B and I by the two hydrogen bond distances Oγ-Thr203···Oη-chromophore and Nδ1-His148···Oη-chromophore. We discuss the characteristics of the I intermediate of wild-type GFP on the bases of the structure estimated from the variant structures by quantum chemical calculations.

Pyrrolizwilline, a unique bacterial alkaloid assembled by a nonribosomal peptide synthetase and non-enzymatic dimerization.

Pyrrolizidine alkaloids (PAs) are a structurally diverse group of heterocyclic specialized metabolites characterized by a core structure comprising a hexahydro-1H-pyrrolizine. PAs are synthesized through two main pathways. In plants, assembly occurs via a homospermidine synthase, and in bacteria, through combined action of a nonribosomal peptide synthetase and a Baeyer-Villiger monooxygenase. While the toxic properties of plant-derived PAs and their prevalence in animal and human foods have been extensively studied, the biological roles and biosynthesis of more complex bacterial PAs are not well understood. Here, we report the identification and characterization of a bacterial biosynthetic gene cluster from Xenorhabdus hominickii, xhpA-G, which is responsible for producing the PA pseudo-dimer pyrrolizwilline. Analysis of X. hominickii promoter exchange mutants together with heterologous expression of xhpA-G in E. coli, revealed a set of pathway intermediates, two of which were chemically synthesized, as well as multiple derivatives. This information was leveraged to propose a detailed biosynthetic pathway to pyrrolizwilline. Furthermore, we have characterized the hydrolase XhpG, the key enzyme in the conversion of the pathway intermediate pyrrolizixenamide to pyrrolizwilline, using X-ray crystallography and small-angle X-ray scattering (SAXS).

Structures of a DNA Polymerase Caught while Incorporating Responsive Dual-Functional Nucleotide Probes.

Functionalizing nucleic acids using DNA polymerases is essential in biophysical and biotechnology applications. This study focuses on understanding how DNA polymerases recognize and incorporate nucleotides with diverse chemical modifications, aiming to develop advanced nucleotide probes. We present the crystal structures of ternary complexes of Thermus aquaticus DNA polymerase (KlenTaq) with C5-heterocycle-modified environment-sensitive 2'-deoxyuridine-5'-triphosphate (dUTP) probes. These nucleotides include SedUTP, BFdUTP and FBFdUTP, which bear selenophene, benzofuran and fluorobenzofuran, respectively, at the C5 position of uracil, and exhibit high conformational sensitivity. SedUTP and FBFdUTP serve as dual-app probes, combining a fluorophore with X-ray anomalous scattering Se or 19F NMR labels. Our study reveals that the size of the heterocycle influences how DNA polymerase families A and B incorporate these modified nucleotides during single nucleotide incorporation and primer extension reactions. Remarkably, FBFdUTP's responsiveness enabled real-time monitoring of the binary complex formation and polymerase activity through fluorescence and 19F NMR. Comparative analysis of incorporation profiles, fluorescence, 19F NMR data, and crystal structures of ternary complexes highlights the enzyme's plasticity. Key insights are provided into the role of gatekeeper amino acids (Arg660 and Arg587) in accommodating and processing these modified substrates, offering a structural basis for next-generation nucleotide probe development.

Expression, purification, and biochemical analysis of the RNA-DNA hybrid helicase Sen1/SETX from Chaetomium thermophilum.

Yeast Sen1 and its vertebrate ortholog Senataxin (also known as SETX) are RNA-DNA resolving helicases. Sen1 and SETX are implicated in multiple critical nuclear functions not limited to but including DNA replication and repair, RNA processing, and transcription. These> 200 kDa helicases have a two-domain architecture with an N-terminal regulatory helical repeat array linked to an SF1b helicase motor core via a variable sized central linker of low complexity sequence. Given the size of these proteins, production of milligram quantities of protein that is suitable for biochemical, biophysical, and protein structural analysis has been challenging. To overcome these limitations, we developed a robust selectable high-yield YFP-fusion protein expression method for Sen1 production in mammalian cells, followed by purification on a high-affinity YFP-binding camelid nanobody support. Herein, we detail methods and protocols for the expression and purification of recombinant Sen1 from the thermophilic fungus Chaetomium thermophilum, and the quantitative characterization of its RNA-DNA duplex resolution activity.