Structural insight into DFMO resistant ornithine decarboxylase from Entamoeba histolytica: an inkling to adaptive evolution

Background: Polyamine biosynthetic pathway is a validated therapeutic target for large number of infectious diseases including cancer, giardiasis and African sleeping sickness, etc. α-Difluoromethylornithine (DFMO), a potent drug used for the treatment of African sleeping sickness is an irreversible inhibitor of ornithine decarboxylase (ODC), the first rate limiting enzyme of polyamine biosynthesis. The enzyme ODC of E. histolytica (EhODC) has been reported to exhibit resistance towards DFMO. Methodology/Principal Finding: The basis for insensitivity towards DFMO was investigated by structural analysis of EhODC and conformational modifications at the active site. Here, we report cloning, purification and crystal structure determination of C-terminal truncated Entamoeba histolytica ornithine decarboxylase (EhODCΔ15). Structure was determined by molecular replacement method and refined to 2.8 Å resolution. The orthorhombic crystal exhibits P212121 symmetry with unit cell parameters a = 76.66, b = 119.28, c = 179.28 Å. Functional as well as evolutionary relations of EhODC with other ODC homologs were predicted on the basis of sequence analysis, phylogeny and structure. Conclusions/Significance: We determined the tetrameric crystal structure of EhODCΔ15, which exists as a dimer in solution. Insensitivity towards DFMO is due to substitution of key substrate binding residues in active site pocket. Additionally, a few more substitutions similar to antizyme inhibitor (AZI), a non-functional homologue of ODCs, were identified in the active site. Here, we establish the fact that EhODC sequence has conserved PLP binding residues; in contrast few substrate binding residues are mutated similar to AZI. Further sequence analysis and structural studies revealed that EhODC may represent as an evolutionary bridge between active decarboxylase and inactive AZI

Biochemical, mutational and in silico structural evidence for a functional dimeric Form of the ornithine decarboxylase from entamoeba histolytica

Background Entamoeba histolytica is responsible for causing amoebiasis. Polyamine biosynthesis pathway enzymes are potential drug targets in parasitic protozoan diseases. The first and rate-limiting step of this pathway is catalyzed by ornithine decarboxylase (ODC). ODC enzyme functions as an obligate dimer. However, partially purified ODC from E. histolytica (EhODC) is reported to exist in a pentameric state. Methodology and Results In present study, the oligomeric state of EhODC was re-investigated. The enzyme was over-expressed in Escherichia coli and purified. Pure protein was used for determination of secondary structure content using circular dichroism spectroscopy. The percentages of α-helix, β-sheets and random coils in EhODC were estimated to be 39%, 25% and 36% respectively. Size-exclusion chromatography and mass spectrophotometry analysis revealed that EhODC enzyme exists in dimeric form. Further, computational model of EhODC dimer was generated. The homodimer contains two separate active sites at the dimer interface with Lys57 and Cys334 residues of opposite monomers contributing to each active site. Molecular dynamic simulations were performed and the dimeric structure was found to be very stable with RMSD value ~0.327 nm. To gain insight into the functional role, the interface residues critical for dimerization and active site formation were identified and mutated. Mutation of Lys57Ala or Cys334Ala completely abolished enzyme activity. Interestingly, partial restoration of the enzyme activity was observed when inactive Lys57Ala and Cys334Ala mutants were mixed confirming that the dimer is the active form. Furthermore, Gly361Tyr and Lys157Ala mutations at the dimer interface were found to abolish the enzyme activity and destabilize the dimer. Conclusion To our knowledge, this is the first report which demonstrates that EhODC is functional in the dimeric form. These findings and availability of 3D structure model of EhODC dimer opens up possibilities for alternate enzyme inhibition strategies by targeting the dimer disruption

Crystal Structure of Aura Virus Capsid Protease and Its Complex with Dioxane: New Insights into Capsid-Glycoprotein Molecular Contacts.

The nucleocapsid core interaction with endodomains of glycoproteins plays a critical role in the alphavirus life cycle that is essential to virus budding. Recent cryo-electron microscopy (cryo-EM) studies provide structural insights into key interactions between capsid protein (CP) and trans-membrane glycoproteins E1 and E2. CP possesses a chymotrypsin-like fold with a hydrophobic pocket at the surface responsible for interaction with glycoproteins. In the present study, crystal structures of the protease domain of CP from Aura virus and its complex with dioxane were determined at 1.81 and 1.98 Å resolution respectively. Due to the absence of crystal structures, homology models of E1 and E2 from Aura virus were generated. The crystal structure of CP and structural models of E1 and E2 were fitted into the cryo-EM density map of Venezuelan equine encephalitis virus (VEEV) for detailed analysis of CP-glycoprotein interactions. Structural analysis revealed that the E2 endodomain consists of a helix-loop-helix motif where the loop region fits into the hydrophobic pocket of CP. Our studies suggest that Cys397, Cys418 and Tyr401 residues of E2 are involved in stabilizing the structure of E2 endodomain. Density map fitting analysis revealed that Pro405, a conserved E2 residue is present in the loop region of the E2 endodomain helix-loop-helix structure and makes intermolecular hydrophobic contacts with the capsid. In the Aura virus capsid protease (AVCP)-dioxane complex structure, dioxane occupies the hydrophobic pocket on CP and structurally mimics the hydrophobic pyrollidine ring of Pro405 in the loop region of E2

Sugarcane bagasse biochar-amended sediment improves growth, survival, and physiological profiles of white-leg shrimp, Litopenaeus vannamei (Boone, 1931) reared in inland saline water

Many parts of globe are confronted with salinization of inland agricultural land and inland saline aquaculture (ISA) pave the way for sustainable production using these resources. Recently, biochar has shown its potential for remediating array of problem related to agriculture; however, studies related to use of biochar in aquaculture is still scarce. Keeping this in prelude, the present study aimed at elucidating the effects of biochar (9 t/ha) amended sediment on overall growth performance and physiological responses on Litopenaeus vannamei reared in inland saline water. A 49-day experiment was performed to evaluate the effects of sugarcane bagasse biochar (SBB) and activated sugarcane bagasse biochar (A-SBB) on water and sediment quality along with growth and health status of L. vannamei. The results of water quality parameters showed a significant increase in K+ and Mg++ with reduction in ammonia-N value in biochar treatments groups. Among the sediment properties, there was a substantially higher water holding capacity, soil organic carbon, pH, and cation exchange capacity in biochar-added treatments in comparison to control. Growth parameters showed a significant increase in weight gain percent, SGR, PER with reduced FCR in biochar-treated groups. Furthermore, the activity of digestive enzymes (protease and amylase), metabolic enzymes (AST, ALT in hepatopancreas), and oxidative stress enzymes (SOD in gills and hepatopancreas; CAT in gills) were significantly higher in biochar-amended treatment groups. The results of the present study revealed biochar-amended sediment has potential to improve vital water and sediment parameters, physiological profiles and growth of L. vannamei juveniles reared in inland saline water; however, future research is needed to demonstrate under usual farming condition

Statistical representation of data collection and structure refinement parameters along with quality of the model accessed by Ramachandran plot.

a<p>value in parentheses are for the highest resolution shell.</p>b<p>R_merge = Σ | <i>I</i>−<i>Ī</i> |/Σ <i>I</i> | where I = observed intensity and Ī = average intensity.</p>c<p>R_free = Σ (|F|_obs−|F|_calc|)/Σ |F|_obs where |F|_obs are observed structure factor amplitudes for a given reflection and |F|_calc are calculated structure factor amplitude.</p

3D structure of <i>Eh</i>ODC monomer.

<p>(A) Cartoon diagram of <i>Eh</i>ODC model generated using Modeller 9v8. (B) Topological arrangement of secondary structures in <i>Eh</i>ODC monomer. Monomer of <i>Eh</i>ODC consists of two domains, β/α-barrel shown in purple and sheet domain having sheet S1 in green, sheet S2 in blue and helices and turns in orange. The helices are presented by circles, strands are represented by triangles and the loops connecting these structures are represented as connecting lines.</p

Superimposition of active site of <i>Eh</i>ODC with <i>Tb</i>ODC bound to DFMO.

<p>Residues of active site at the dimer interface are represented in sticks. <i>Tb</i>ODC residues are colored with green, <i>Eh</i>ODC residues are colored with orange. PLP and DFMO are colored with blue and polar interactions were indicated by black dashes; water molecule in shown in red sphere. Residues with (′) symbol are of opposite monomer.</p

Schematic representation of homodimers and heterodimer in the mixture of <i>Eh</i>ODC Cys334Ala and Lys57Ala mutants.

<p>(A–C) Homodimer formation of wild-type and mutants of <i>Eh</i>ODC in individual solutions. (D) Possible combinations of <i>Eh</i>ODC monomeric subunits in the mixture of Cys334Ala and Lys57Ala mutants forming heterodimer and homodimers.</p

Purification and molecular mass determination of <i>Eh</i>ODC.

<p>(A) Affinity purification of <i>Eh</i>ODC showing purified protein in 12% SDS-PAGE. Lane 1: Molecular weight marker; Lane 2: Purified <i>Eh</i>ODC-His tagged protein; Lane 3: Purified His tag cleaved protein with molecular weight ∼46 kDa. (B) Size-exclusion chromatography profile of <i>Eh</i>ODC and 12% SDS-PAGE (insert) analysis of major peak fractions. (C) The elution profile of standard molecular weight markers from size exclusion chromatography through HiLoad 16/60 Superdex 200 column. The column void volume (V_o) and molecular weight (kDa) of standard proteins are indicated.</p