Crystal structure of pyrrolizidine alkaloid N-oxygenase from the grasshopper 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.

Download Full-text

pH-Dependent Thermal Stability of Vibrio cholerae L-asparaginase

Protein and Peptide Letters ◽

10.2174/0929866526666190617092944 ◽

2019 ◽

Vol 26 (10) ◽

pp. 743-750 ◽

Cited By ~ 1

Author(s):

Remya Radha ◽

Sathyanarayana N. Gummadi

Keyword(s):

Vibrio Cholerae ◽

Conformational Changes ◽

Kinetic Studies ◽

Structural Features ◽

Structure Stability ◽

Kcal Mole ◽

Scanning Calorimetry ◽

Wide Range ◽

Ph Dependent ◽

Ph Range

Background:pH is one of the decisive macromolecular properties of proteins that significantly affects enzyme structure, stability and reaction rate. Change in pH may protonate or deprotonate the side group of aminoacid residues in the protein, thereby resulting in changes in chemical and structural features. Hence studies on the kinetics of enzyme deactivation by pH are important for assessing the bio-functionality of industrial enzymes. L-asparaginase is one such important enzyme that has potent applications in cancer therapy and food industry.Objective:The objective of the study is to understand and analyze the influence of pH on deactivation and stability of Vibrio cholerae L-asparaginase.Methods:Kinetic studies were conducted to analyze the effect of pH on stability and deactivation of Vibrio cholerae L-asparaginase. Circular Dichroism (CD) and Differential Scanning Calorimetry (DSC) studies have been carried out to understand the pH-dependent conformational changes in the secondary structure of V. cholerae L-asparaginase.Results:The enzyme was found to be least stable at extreme acidic conditions (pH< 4.5) and exhibited a gradual increase in melting temperature from 40 to 81 °C within pH range of 4.0 to 7.0. Thermodynamic properties of protein were estimated and at pH 7.0 the protein exhibited ΔG37of 26.31 kcal mole-1, ΔH of 204.27 kcal mole-1 and ΔS of 574.06 cal mole-1 K-1.Conclusion:The stability and thermodynamic analysis revealed that V. cholerae L-asparaginase was highly stable over a wide range of pH, with the highest stability in the pH range of 5.0–7.0.

Download Full-text

Serum Cholinesterase Variants: Examination of Several Differential Inhibitors, Salts, and Buffers Used to Measure Enzyme Activity

Clinical Chemistry ◽

10.1093/clinchem/17.3.183 ◽

1971 ◽

Vol 17 (3) ◽

pp. 183-191 ◽

Cited By ~ 30

Author(s):

Philip J Garry

Keyword(s):

Enzyme Activity ◽

Sodium Fluoride ◽

Phosphate Buffer ◽

Divalent Cations ◽

Kinetic Studies ◽

Competitive Inhibitor ◽

Serum Cholinesterase ◽

Low Concentrations

Abstract Dibucaine, used as a differential inhibitor with acetyl-, propionyl-, and butyrylthiocholine as substrate, clearly identified the "usual" and "atypical" serum cholinesterases. Succinylcholine was also used successfully as a differential inhibitor with butyrylthiocholine as substrate. Sodium fluoride, used as a differential inhibitor, gave conflicting results, depending on whether Tris or phosphate buffer was used in the assay. Mono- and divalent cations (NaCl, KCl, MgCl2, CaCl2, and BaCl2) activated the "usual" and inhibited the "atypical" enzyme at low concentrations. The "usual" enzyme had the same activity in 0.05 mol of Tris or phosphate buffer per liter, while the heterozygous and "atypical" enzymes showed 12 and 42% inhibition, respectively, when assayed in the phosphate buffer. Kinetic studies showed the phosphate acted as a competitive inhibitor of "atypical" enzyme. Km values, determined for "usual" and "atypical" enzymes, were 0.057 and 0.226 mmol/liter, respectively, with butyrylthiocholine as substrate.

Download Full-text

Fluorescence quenching and kinetic studies of conformational changes induced by DNA and cAMP binding to cAMP receptor protein from Escherichia coli

FEBS Journal ◽

10.1111/j.1742-4658.2005.04540.x ◽

2005 ◽

Vol 272 (5) ◽

pp. 1103-1116 ◽

Cited By ~ 8

Author(s):

Magdalena Tworzydło ◽

Agnieszka Polit ◽

Jan Mikołajczak ◽

Zygmunt Wasylewski

Keyword(s):

Escherichia Coli ◽

Fluorescence Quenching ◽

Conformational Changes ◽

Kinetic Studies ◽

Receptor Protein ◽

Camp Receptor Protein ◽

Camp Receptor

Download Full-text

Study of Extraction and Enzymatic Properties of Cell-Envelope Proteinases from a Novel Wild Lactobacillus plantarum LP69

Catalysts ◽

10.3390/catal8080325 ◽

2018 ◽

Vol 8 (8) ◽

pp. 325 ◽

Cited By ~ 1

Author(s):

He Chen ◽

Jie Huang ◽

Binyun Cao ◽

Li Chen ◽

Na Song ◽

...

Keyword(s):

Enzyme Activity ◽

Lactobacillus Plantarum ◽

Industrial Development ◽

Extraction Efficiency ◽

Specific Activity ◽

Cell Envelope ◽

Serine Proteinase ◽

Kinetic Studies ◽

Predicted Values ◽

Hydrolyze Whey Protein

Lactobacilli cell-envelope proteinases (CEPs) have been widely used in the development of new streams of blockbuster nutraceuticals because of numerous biopharmaceutical potentials; thus, the development of viable methods for CEP extraction and the improvement of extraction efficiency will promote their full-scale application. In this study, CEP from a novel wild Lactobacillus plantarum LP69 was released from cells by incubating in calcium-free buffer. The extraction conditions of CEP were optimized by response surface methodology with the enzyme activity and specific activity as the detective marker. The optimal extraction conditions were: time of 80 min, temperature of 39 °C and buffer pH of 6.5. Under these conditions, enzyme activity and specific activity were (23.94 ± 0.86) U/mL and (1.37 ± 0.03) U/mg, respectively, which were well matched with the predicted values (22.12 U/mL and 1.36 U/mg). Optimal activity of the crude CEP occurred at pH 8.0 and 40 °C. It is a metallopeptidase, activated by Ca2+, inhibited by Zn2+ and ethylene-diamine-tetra-acetic acid, and a serine proteinase which is inhibited by phenylmethylsulfonyl fluoride. Kinetic studies showed that CEP from LP69 could hydrolyze whey protein, lactoglobulin and casein. Our study improves the extraction efficiency of CEPs from LP69, providing the reference for their industrial development.

Download Full-text

The first crystal structure of the peptidase domain of the U32 peptidase family

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004715019549 ◽

2015 ◽

Vol 71 (12) ◽

pp. 2505-2512 ◽

Cited By ~ 4

Author(s):

Magdalena Schacherl ◽

Angelika A. M. Montada ◽

Elena Brunstein ◽

Ulrich Baumann

Keyword(s):

Crystal Structure ◽

Catalytic Mechanism ◽

Quaternary Structure ◽

Catalytic Domain ◽

Three Dimensional ◽

Zinc Ion ◽

Crystal Structure Analysis ◽

Dimensional Structure ◽

Sequence Motifs ◽

Conserved Sequence

The U32 family is a collection of over 2500 annotated peptidases in the MEROPS database with unknown catalytic mechanism. They mainly occur in bacteria and archaea, but a few representatives have also been identified in eukarya. Many of the U32 members have been linked to pathogenicity, such as proteins fromHelicobacterandSalmonella. The first crystal structure analysis of a U32 catalytic domain fromMethanopyrus kandleri(genemk0906) reveals a modified (βα)8TIM-barrel fold with some unique features. The connecting segment between strands β7 and β8 is extended and helix α7 is located on top of the C-terminal end of the barrel body. The protein exhibits a dimeric quaternary structure in which a zinc ion is symmetrically bound by histidine and cysteine side chains from both monomers. These residues reside in conserved sequence motifs. No typical proteolytic motifs are discernible in the three-dimensional structure, and biochemical assays failed to demonstrate proteolytic activity. A tunnel in which an acetate ion is bound is located in the C-terminal part of the β-barrel. Two hydrophobic grooves lead to a tunnel at the C-terminal end of the barrel in which an acetate ion is bound. One of the grooves binds to aStrep-Tag II of another dimer in the crystal lattice. Thus, these grooves may be binding sites for hydrophobic peptides or other ligands.

Download Full-text

Kinetic aspects of conformational changes in proteins. I. Rate of regain of enzyme activity from denatured proteins

Biochemistry ◽

10.1021/bi00781a011 ◽

1971 ◽

Vol 10 (5) ◽

pp. 792-798 ◽

Cited By ~ 131

Author(s):

John W. Teipel ◽

Daniel E. Koshland

Keyword(s):

Enzyme Activity ◽

Conformational Changes ◽

Denatured Proteins

Download Full-text

A new three-dimensional anionic cadmium(II) dicyanamide network

Acta Crystallographica Section C Structural Chemistry ◽

10.1107/s2053229614022360 ◽

2014 ◽

Vol 70 (11) ◽

pp. 1054-1056 ◽

Cited By ~ 9

Author(s):

Qiang Li ◽

Hui-Ting Wang

Keyword(s):

Crystal Structure ◽

Aqueous Solution ◽

Unit Cell Volume ◽

Three Dimensional ◽

Building Blocks ◽

The Other ◽

Dimensional Structure ◽

Cadmium Nitrate ◽

Nitrate Tetrahydrate ◽

Anionic Framework

A new cadmium dicyanamide complex, poly[tetramethylphosphonium [μ-chlorido-di-μ-dicyanamido-κ4N1:N5-cadmium(II)]], [(CH3)4P][Cd(NCNCN)2Cl], was synthesized by the reaction of tetramethylphosphonium chloride, cadmium nitrate tetrahydrate and sodium dicyanamide in aqueous solution. In the crystal structure, each CdIIatom is octahedrally coordinated by four terminal N atoms from four anionic dicyanamide (dca) ligands and by two chloride ligands. The dicyanamide ligands play two different roles in the building up of the structure; one role results in the formation of [Cd(dca)Cl]2building blocks, while the other links the building blocks into a three-dimensional structure. The anionic framework exhibits a solvent-accessible void of 673.8 Å3, amounting to 47.44% of the total unit-cell volume. The cavities in the network are occupied by pairs of tetramethylphosphonium cations.

Download Full-text

Crystal structure of 2-benzylamino-4-(4-methoxyphenyl)-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridine-3-carbonitrile

Acta Crystallographica Section E Structure Reports Online ◽

10.1107/s1600536814024878 ◽

2014 ◽

Vol 70 (12) ◽

pp. 525-527 ◽

Cited By ~ 1

Author(s):

R. A. Nagalakshmi ◽

J. Suresh ◽

S. Maharani ◽

R. Ranjith Kumar ◽

P. L. Nilantha Lakshman

Keyword(s):

Crystal Structure ◽

Pyridine Ring ◽

Three Dimensional ◽

Chair Conformation ◽

Dimensional Structure ◽

Compound C ◽

Centroid Distance ◽

Steric Crowding ◽

Benzene Rings ◽

Group A

The title compound, C25H25N3O, comprises a 2-aminopyridine ring fused with a cycloheptane ring, which adopts a chair conformation. The central pyridine ring (r.m.s. deviation = 0.013 Å) carries three substituents,viz.a benzylamino group, a methoxyphenyl ring and a carbonitrile group. The N atom of the carbonitrile group is significantly displaced [by 0.2247 (1) Å] from the plane of the pyridine ring, probably due to steric crowding involving the adjacent substituents. The phenyl and benzene rings are inclined to one another by 58.91 (7)° and to the pyridine ring by 76.68 (7) and 49.80 (6)°, respectively. In the crystal, inversion dimers linked by pairs of N—H...Nnitrilehydrogen bonds generateR22(14) loops. The dimers are linked by C—H...π and slipped parallel π–π interactions [centroid–centroid distance = 3.6532 (3) Å] into a three-dimensional structure.

Download Full-text