Structural insights into the calcium dependence of Stig cyclases

The hPrp19–CDC5L complex plays a crucial role during human pre-mRNA splicing by catalytic activation of the spliceosome. In order to elucidate the molecular architecture of the hPrp19–CDC5L complex, the crystal structure of CTNNBL1, one of the major components of this complex, was determined. Unlike canonical ARM-repeat proteins such as β-catenin and importin-α, CTNNBL1 was found to contain a twisted and extended ARM-repeat structure at the C-terminal domain and, more importantly, the protein formed a stable dimer. A highly negatively charged patch formed in the N-terminal ARM-repeat domain of CTNNBL1 provides a binding site for CDC5L, a binding partner of the protein in the hPrp19–CDC5L complex, and these two proteins form a complex with a stoichiometry of 2:2. These findings not only present the crystal structure of a novel ARM-repeat protein, CTNNBL1, but also provide insights into the detailed molecular architecture of the hPrp19–CDC5L complex.

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Structural insights into the substrate specificity of a glycoside hydrolase family 5 lichenase from Caldicellulosiruptor sp. F32

Biochemical Journal ◽

10.1042/bcj20170328 ◽

2017 ◽

Vol 474 (20) ◽

pp. 3373-3389 ◽

Cited By ~ 9

Author(s):

Dong-Dong Meng ◽

Xi Liu ◽

Sheng Dong ◽

Ye-Fei Wang ◽

Xiao-Qing Ma ◽

...

Keyword(s):

Crystal Structure ◽

Substrate Specificity ◽

Glycoside Hydrolase ◽

Complex Structure ◽

Dynamics Simulation ◽

Structural Basis ◽

Glycoside Hydrolase Family ◽

Substrate Selectivity ◽

Glucose Residue ◽

Structural Insights

Glycoside hydrolase (GH) family 5 is one of the largest GH families with various GH activities including lichenase, but the structural basis of the GH5 lichenase activity is still unknown. A novel thermostable lichenase F32EG5 belonging to GH5 was identified from an extremely thermophilic bacterium Caldicellulosiruptor sp. F32. F32EG5 is a bi-functional cellulose and a lichenan-degrading enzyme, and exhibited a high activity on β-1,3-1,4-glucan but side activity on cellulose. Thin-layer chromatography and NMR analyses indicated that F32EG5 cleaved the β-1,4 linkage or the β-1,3 linkage while a 4-O-substitued glucose residue linked to a glucose residue through a β-1,3 linkage, which is completely different from extensively studied GH16 lichenase that catalyses strict endo-hydrolysis of the β-1,4-glycosidic linkage adjacent to a 3-O-substitued glucose residue in the mixed-linked β-glucans. The crystal structure of F32EG5 was determined to 2.8 Å resolution, and the crystal structure of the complex of F32EG5 E193Q mutant and cellotetraose was determined to 1.7 Å resolution, which revealed that the exit subsites of substrate-binding sites contribute to both thermostability and substrate specificity of F32EG5. The sugar chain showed a sharp bend in the complex structure, suggesting that a substrate cleft fitting to the bent sugar chains in lichenan is a common feature of GH5 lichenases. The mechanism of thermostability and substrate selectivity of F32EG5 was further demonstrated by molecular dynamics simulation and site-directed mutagenesis. These results provide biochemical and structural insights into thermostability and substrate selectivity of GH5 lichenases, which have potential in industrial processes.

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Structural and biochemical characterization of bifunctional XynA

10.1101/2020.10.20.348094 ◽

2020 ◽

Author(s):

Wei Xie ◽

Qi Yu ◽

Yun Liu ◽

Ruoting Cao ◽

Ruiqing Zhang ◽

...

Keyword(s):

Crystal Structure ◽

Enzyme Activity ◽

Catalytic Mechanism ◽

Biochemical Characterization ◽

Cellulase Activity ◽

Cost Savings ◽

Site Directed Mutagenesis ◽

Enzyme Activity Assay ◽

Great Cost ◽

Terminal Domain

AbstractXylan and cellulose are the two major constituents in numerous types of lignocellulosic biomass, representing a promising resource for biofuels and other biobased industries. The efficient degradation of lignocellulose requires the synergistic actions of cellulase and xylanase. Thus, bifunctional enzyme incorporated xylanase/cellulase activity has attracted considerable attention since it has great cost savings potential. Recently, a novel GH10 family enzyme XynA identified from Bacillus sp. is found to degrade both cellulose and xylan. To understand its molecular catalytic mechanism, here we first solve the crystal structure of XynA at 2.3 Å. XynA is characterized with a classic (α/β)8 TIM-barrel fold (GH10 domain) flanked by the flexible N-terminal domain and C-terminal domain. Circular dichroism, protein thermal shift and enzyme activity assays reveal that conserved residues Glu182 and Glu280 are both important for catalytic activities of XynA, which is verified by the crystal structure of XynA with E182A/E280A double mutant. Molecular docking studies of XynA with xylohexaose and cellohexaose as well as site-directed mutagenesis and enzyme activity assay demonstrat that Gln250 and His252 are indispensible to cellulase and bifunctional activity, separately. These results elucidate the structural and biochemical features of XynA, providing clues for further modification of XynA for industrial application.

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Crystal structure of pyrrolizidine alkaloid N-oxygenase from the grasshopper Zonocerus variegatus

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798318003510 ◽

2018 ◽

Vol 74 (5) ◽

pp. 422-432 ◽

Cited By ~ 5

Author(s):

Christian Kubitza ◽

Annette Faust ◽

Miriam Gutt ◽

Luzia Gäth ◽

Dietrich Ober ◽

...

Keyword(s):

Crystal Structure ◽

Enzyme Activity ◽

Conformational Changes ◽

Pyrrolizidine Alkaloid ◽

Chemical Defence ◽

Kinetic Studies ◽

Food Plants ◽

Single Amino Acid ◽

Dimensional Structure ◽

Zonocerus Variegatus

The high-resolution crystal structure of the flavin-dependent monooxygenase (FMO) from the African locust Zonocerus variegatus is presented and the kinetics of structure-based protein variants are discussed. Z. variegatus expresses three flavin-dependent monooxygenase (ZvFMO) isoforms which contribute to a counterstrategy against pyrrolizidine alkaloids (PAs). PAs are protoxic compounds produced by some angiosperm lineages as a chemical defence against herbivores. N-Oxygenation of PAs and the accumulation of PA N-oxides within their haemolymph result in two evolutionary advantages for these insects: (i) they circumvent the defence mechanism of their food plants and (ii) they can use PA N-oxides to protect themselves against predators, which cannot cope with the toxic PAs. Despite a high degree of sequence identity and a similar substrate spectrum, the three ZvFMO isoforms differ greatly in enzyme activity. Here, the crystal structure of the Z. variegatus PA N-oxygenase (ZvPNO), the most active ZvFMO isoform, is reported at 1.6 Å resolution together with kinetic studies of a second isoform, ZvFMOa. This is the first available crystal structure of an FMO from class B (of six different FMO subclasses, A–F) within the family of flavin-dependent monooxygenases that originates from a more highly developed organism than yeast. Despite the differences in sequence between family members, their overall structure is very similar. This indicates the need for high conservation of the three-dimensional structure for this type of reaction throughout all kingdoms of life. Nevertheless, this structure provides the closest relative to the human enzyme that is currently available for modelling studies. Of note, the crystal structure of ZvPNO reveals a unique dimeric arrangement as well as small conformational changes within the active site that have not been observed before. A newly observed kink within helix α8 close to the substrate-binding path might indicate a potential mechanism for product release. The data show that even single amino-acid exchanges in the substrate-entry path, rather than the binding site, have a significant impact on the specific enzyme activity of the isoforms.

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Design and Synthesis of N-phenyl Phthalimides as Potent Protoporphyrinogen Oxidase Inhibitors

Molecules ◽

10.3390/molecules24234363 ◽

2019 ◽

Vol 24 (23) ◽

pp. 4363

Author(s):

Wei Gao ◽

Xiaotian Li ◽

Da Ren ◽

Susu Sun ◽

Jingqian Huo ◽

...

Keyword(s):

Crystal Structure ◽

Enzyme Activity ◽

Molecular Docking ◽

Molecular Design ◽

Design Strategy ◽

Docking Studies ◽

Activity Assay ◽

Enzyme Activity Assay ◽

Protoporphyrinogen Oxidase ◽

Design And Synthesis

Protoporphyrinogen oxidase (PPO) has been identified as one of the most promising targets for herbicide discovery. A series of novel phthalimide derivatives were designed by molecular docking studies targeting the crystal structure of mitochondrial PPO from tobacco (mtPPO, PDB: 1SEZ) by using Flumioxazin as a lead, after which the derivatives were synthesized and characterized, and their herbicidal activities were subsequently evaluated. The herbicidal bioassay results showed that compounds such as 3a (2-(4-bromo-2,6-difluorophenyl) isoindoline-1,3-dione), 3d (methyl 2-(4-chloro-1,3-dioxoisoindolin-2-yl)-5-fluorobenzoate), 3g (4-chloro-2-(5-methylisoxazol-3-yl) isoindoline-1,3-dione), 3j (4-chloro-2-(thiophen-2-ylmethyl) isoindoline-1,3-dione) and 3r (2-(4-bromo-2,6-difluorophenyl)-4-fluoroisoindoline-1,3-dione) had good herbicidal activities; among them, 3a showed excellent herbicidal efficacy against A. retroflexus and B. campestris via the small cup method and via pre-emergence and post-emergence spray treatments. The efficacy was comparable to that of the commercial herbicides Flumioxazin, Atrazine, and Chlortoluron. Further, the enzyme activity assay results suggest that the mode of action of compound 3a involves the inhibition of the PPO enzyme, and 3a showed better inhibitory activity against PPO than did Flumioxazin. These results indicate that our molecular design strategy contributes to the development of novel promising PPO inhibitors.

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Structure–function studies of human deoxyhypusine synthase: identification of amino acid residues critical for the binding of spermidine and NAD

Biochemical Journal ◽

10.1042/bj3550841 ◽

2001 ◽

Vol 355 (3) ◽

pp. 841-849 ◽

Cited By ~ 21

Author(s):

Chang Hoon LEE ◽

Patrick Y. UM ◽

Myung Hee PARK

Keyword(s):

Crystal Structure ◽

Enzyme Activity ◽

Amino Acid ◽

Binding Sites ◽

Binding Pocket ◽

Complete Loss ◽

Amino Acid Residues ◽

Deoxyhypusine Synthase ◽

Positive Identification ◽

Insight Into

Deoxyhypusine synthase catalyses the first step in the biosynthesis of hypusine [Nε-(4-amino-2-hydroxybutyl)lysine]. The crystal structure of human deoxyhypusine synthase in complex with NAD revealed four NAD-binding sites per enzyme tetramer, and led to a prediction of the spermidine-binding pocket. We have replaced each of the seven amino acid residues at the predicted spermidine-binding site, and eleven residues that contact NAD, on an individual basis with alanine. Of the amino acid residues at the spermidine site, substitution of Asp-243, Trp-327, His-288, Asp-316 or Glu-323 with alanine caused an almost complete loss of spermidine binding and enzyme activity; only the mutation Tyr-305 → Ala showed partial binding and activity. His-288 → Ala was also deficient in terms of binding NAD. NAD binding was significantly reduced in all of the NAD-site mutant enzymes, except for Glu-137 → Ala, which showed a normal binding of NAD, but was totally lacking in spermidine binding. Of the NAD-site mutant enzymes, Asp-342 → Ala, Asp-313 → Ala and Asp-238 → Ala displayed the lowest binding of NAD. These enzymes and His-288Ala also showed a reduced binding of spermidine, presumably because spermidine binding is dependent on NAD. These findings permit the positive identification of amino acid residues critical for binding of spermidine and NAD, and provide a new insight into the complex molecular interactions involved in the deoxyhypusine synthase reaction.

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Structural insights into DNA repair by RNase T

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314097915 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C208-C208

Author(s):

Yu-Yuan Hsiao ◽

Hanna Yuan

Keyword(s):

Crystal Structure ◽

Dna Repair ◽

Binding Mode ◽

Heteroduplex Dna ◽

Dna Enzymes ◽

Endonuclease V ◽

Dna Damaging Agents ◽

Bulge Dna ◽

Repair Pathways ◽

Structural Insights

DNA repair is generally accomplished by a coordinated effort via several types of DNA enzymes, including endonucleases, exonucleases, helicases, polymerases and ligases. Among all these DNA enzymes, the molecular functions of exonucleases, which bind at the 3′ or 5′ end of DNA and cleave one nucleotide at a time, are least understood in how they select DNA substrates for binding and trimming. Here we show that the DEDDh family exonuclease RNase T is critical for Escherichia coli resistance to various DNA damaging agents and UV radiation. RNase T specifically trims the 3′ end of structured DNA, including bulge, bubble and Y-structured DNA, and it can work with Endonuclease V to restore the deaminated base in an inosine-containing heteroduplex DNA. Our crystal structure analyses further reveal how RNase T recognizes the bulge DNA by inserting a phenylalanine into the bulge, and as a result the 3′ end of blunt-end bulge DNA can be digested by RNase T. In contrast, the homodimeric RNase T interacts with the Y-structured DNA by a different binding mode via a single protomer so that the 3′ overhang of the Y-structured DNA can be trimmed closely to the duplex region. Our data suggest that RNase T likely processes bulge and bubble DNA in the Endonuclease V-dependent DNA repair, whereas it processes Y-structured DNA in UV-induced and various other DNA repair pathways. This study thus provides mechanistic insights for RNase T and thousands of DEDDh-family exonucleases in DNA 3′-end processing.

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Structural insights into species-specific features of the ribosome from the pathogen Staphylococcus aureus

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1517952112 ◽

2015 ◽

Vol 112 (43) ◽

pp. E5805-E5814 ◽

Cited By ~ 68

Author(s):

Zohar Eyal ◽

Donna Matzov ◽

Miri Krupkin ◽

Itai Wekselman ◽

Susanne Paukner ◽

...

Keyword(s):

Crystal Structure ◽

Staphylococcus Aureus ◽

Ribosomal Subunit ◽

Binding Modes ◽

Large Ribosomal Subunit ◽

Structural Motifs ◽

Antibiotic Drugs ◽

Species Specific ◽

Structural Insights ◽

Shed Light

The emergence of bacterial multidrug resistance to antibiotics threatens to cause regression to the preantibiotic era. Here we present the crystal structure of the large ribosomal subunit from Staphylococcus aureus, a versatile Gram-positive aggressive pathogen, and its complexes with the known antibiotics linezolid and telithromycin, as well as with a new, highly potent pleuromutilin derivative, BC-3205. These crystal structures shed light on specific structural motifs of the S. aureus ribosome and the binding modes of the aforementioned antibiotics. Moreover, by analyzing the ribosome structure and comparing it with those of nonpathogenic bacterial models, we identified some unique internal and peripheral structural motifs that may be potential candidates for improving known antibiotics and for use in the design of selective antibiotic drugs against S. aureus.

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