Crystal structure of α-amylase fromOryza sativa: molecular insights into enzyme activity and thermostability

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 the calcium dependence of Stig cyclases

RSC Advances ◽

10.1039/c9ra00960d ◽

2019 ◽

Vol 9 (23) ◽

pp. 13182-13185 ◽

Cited By ~ 2

Author(s):

Xueke Tang ◽

Jing Xue ◽

Yunyun Yang ◽

Tzu-Ping Ko ◽

Chin-Yu Chen ◽

...

Keyword(s):

Crystal Structure ◽

Enzyme Activity ◽

Calcium Dependence ◽

Structural Insights

Crystal structure of a calcium-depleted Stig cyclase revealing the mechanism of metal-dependence of enzyme activity.

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Apple 1-Aminocyclopropane-1-carboxylate Synthase in Complex with the Inhibitor l-Aminoethoxyvinylglycine

Journal of Biological Chemistry ◽

10.1074/jbc.m208427200 ◽

2002 ◽

Vol 277 (51) ◽

pp. 49735-49742 ◽

Cited By ~ 32

Author(s):

Guido Capitani ◽

Darla L. McCarthy ◽

Heinz Gut ◽

Markus G. Grütter ◽

Jack F. Kirsch

Keyword(s):

Crystal Structure ◽

Plant Physiology ◽

Enzyme Activity ◽

Rate Constant ◽

Plant Hormone ◽

Potent Inhibitor ◽

Acc Synthase ◽

Dissociation Rate ◽

Stopped Flow ◽

Structural Assignment

The 1.6-Å crystal structure of the covalent ketimine complex of apple 1-aminocyclopropane-1-carboxylate (ACC) synthase with the potent inhibitorl-aminoethoxyvinylglycine (AVG) is described. ACC synthase catalyzes the committed step in the biosynthesis of ethylene, a plant hormone that is responsible for the initiation of fruit ripening and for regulating many other developmental processes. AVG is widely used in plant physiology studies to inhibit the activity of ACC synthase. The structural assignment is supported by the fact that the complex absorbs maximally at 341 nm. These results are not in accord with the recently reported crystal structure of the tomato ACC synthase AVG complex, which claims that the inhibitor only associates noncovalently. The rate constant for the association of AVG with apple ACC synthase was determined by stopped-flow spectrophotometry (2.1 × 105m−1s−1) and by the rate of loss of enzyme activity (1.1 × 105m−1s−1). The dissociation rate constant determined by activity recovery is 2.4 × 10−6s−1. Thus, the calculatedKdvalue is 10–20 pm.

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Characterization and crystal structure of a novel zearalenone hydrolase fromCladophialophora bantiana

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x17011840 ◽

2017 ◽

Vol 73 (9) ◽

pp. 515-519 ◽

Cited By ~ 8

Author(s):

Renjie Hui ◽

Xiangying Hu ◽

Wenting Liu ◽

Weidong Liu ◽

Yingying Zheng ◽

...

Keyword(s):

Crystal Structure ◽

Heat Treatment ◽

Enzyme Activity ◽

Active Site ◽

Animal Feed ◽

Hydrolytic Degradation ◽

Catalytic Triad ◽

Economic Losses ◽

Clonostachys Rosea ◽

Detoxifying Enzyme

Zearalenone (ZEN) is a mycotoxin which causes huge economic losses in the food and animal feed industries. The lactonase ZHD101 fromClonostachys rosea, which catalyzes the hydrolytic degradation of ZEN, is the only known ZEN-detoxifying enzyme. Here, a protein homologous to ZHD101, denoted CbZHD, fromCladophialophora batianawas expressed and characterized. Sequence alignment indicates that CbZHD possesses the same catalytic triad and ZEN-interacting residues as found in ZHD101. CbZHD exhibits optimal enzyme activity at 35°C and pH 8, and is sensitive to heat treatment. The crystal structure of apo CbZHD was determined to 1.75 Å resolution. The active-site compositions of CbZHD and ZHD101 were analyzed.

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