title: Structural insight into DFMO resistant ornithine decarboxylase from Entamoeba histolytica: an inkling to adaptive evolution.
abstract: 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.
  
  
  RESULTS
  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 P2(1)2(1)2(1) 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
  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.
authors: Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, India.
authors: Preeti
authors: Satya
authors: S
authors: Tapas
authors: Pravindra
authors: P
authors: Kumar
authors: Rentala
authors: R
authors: Madhubala
authors: Shailly
authors: S
authors: Tomar
mesh_terms: D000222:Adaptation, Physiological; D000595:Amino Acid Sequence; D005075:Biological Evolution; D020134:Catalytic Domain; D002850:Chromatography, Gel; D018360:Crystallography, X-Ray; D000518:Eflornithine / Q000737:chemistry / Q000494:pharmacology*; D004748:Entamoeba histolytica / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009955:Ornithine Decarboxylase / Q000737:chemistry / Q000302:isolation & purification; D065108:Ornithine Decarboxylase Inhibitors; D010802:Phylogeny; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry; D011506:Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship
chemical_list: D004791:Enzyme Inhibitors; D065108:Ornithine Decarboxylase Inhibitors; D021122:Protein Subunits; D011506:Proteins; C031017:ornithine decarboxylase antizyme; D009955:Ornithine Decarboxylase; D000518:Eflornithine