title: Active site mapping of Loxosceles phospholipases D: Biochemical and biological features.
abstract: Brown spider phospholipases D from Loxosceles venoms are among the most widely studied toxins since they induce dermonecrosis, triggering inflammatory responses, increase vascular permeability, cause hemolysis, and renal failure. The catalytic (H12 and H47) and metal-ion binding (E32 and D34) residues in Loxosceles intermedia phospholipase D (LiRecDT1) were mutated to understand their roles in the observed activities. All mutants were identified using whole venom serum antibodies and a specific antibody to wild-type LiRecDT1, they were also analyzed by circular dichroism (CD) and differential scanning calorimetry (DSC). The phospholipase D activities of H12A, H47A, H12A-H47A, E32, D34 and E32A-D34A, such as vascular permeability, dermonecrosis, and hemolytic effects were inhibited. The mutant Y228A was equally detrimental to biochemical and biological effects of phospholipase D, suggesting an essential role of this residue in substrate recognition and binding. On the other hand, the mutant C53A-C201A reduced the enzyme's ability to hydrolyze phospholipids and promote dermonecrosis, hemolytic, and vascular effects. These results provide the basis understanding the importance of specific residues in the observed activities and contribute to the design of synthetic and specific inhibitors for Brown spider venom phospholipases D.
authors.affiliation: Department of Cell Biology, Federal University of Paraná (UFPR), Curitiba, PR, Brazil.
authors.forename: L
authors.initials: L
authors.lastname: Vuitika
authors.affiliation: Department of Cell Biology, Federal University of Paraná (UFPR), Curitiba, PR, Brazil.
authors.forename: D
authors.initials: D
authors.lastname: Chaves-Moreira
authors.affiliation: Multiuser Center for Biomolecular Innovation, Department of Physics, São Paulo State University (UNESP), São José do Rio Preto, SP, Brazil.
authors.forename: I
authors.initials: I
authors.lastname: Caruso
authors.affiliation: Department of Biochemistry, Federal University of São Paulo (UNIFESP), São Paulo, SP, Brazil.
authors.forename: M A
authors.initials: MA
authors.lastname: Lima
authors.affiliation: Department of Cell Biology, Federal University of Paraná (UFPR), Curitiba, PR, Brazil.
authors.forename: F H
authors.initials: FH
authors.lastname: Matsubara
authors.affiliation: Brazilian Biosciences National Laboratory (LNBio), National Center for Research in Energy and Materials (CNPEM), Campinas, SP, Brazil.
authors.forename: M T
authors.initials: MT
authors.lastname: Murakami
authors.affiliation: Department of Biochemistry, Federal University of São Paulo (UNIFESP), São Paulo, SP, Brazil.
authors.forename: H K
authors.initials: HK
authors.lastname: Takahashi
authors.affiliation: Department of Biochemistry, Federal University of São Paulo (UNIFESP), São Paulo, SP, Brazil.
authors.forename: M S
authors.initials: MS
authors.lastname: Toledo
authors.affiliation: Multiuser Center for Biomolecular Innovation, Department of Physics, São Paulo State University (UNESP), São José do Rio Preto, SP, Brazil.
authors.forename: M A
authors.initials: MA
authors.lastname: Coronado
authors.affiliation: Department of Biochemistry, Federal University of São Paulo (UNIFESP), São Paulo, SP, Brazil.
authors.forename: H B
authors.initials: HB
authors.lastname: Nader
authors.affiliation: Department of Cell Biology, Federal University of Paraná (UFPR), Curitiba, PR, Brazil.
authors.forename: A
authors.initials: A
authors.lastname: Senff-Ribeiro
authors.affiliation: Department of Cell Biology, Federal University of Paraná (UFPR), Curitiba, PR, Brazil.
authors.forename: O M
authors.initials: OM
authors.lastname: Chaim
authors.affiliation: Multiuser Center for Biomolecular Innovation, Department of Physics, São Paulo State University (UNESP), São José do Rio Preto, SP, Brazil.
authors.forename: R K
authors.initials: RK
authors.lastname: Arni
authors.affiliation: Department of Cell Biology, Federal University of Paraná (UFPR), Curitiba, PR, Brazil. Electronic address: veigass@ufpr.br.
authors.forename: S S
authors.initials: SS
authors.lastname: Veiga
mesh_terms: D000818:Animals; D063287:Brown Recluse Spider / Q000737:chemistry / Q000201:enzymology; D002199:Capillary Permeability; D020134:Catalytic Domain / Q000235:genetics*; D002942:Circular Dichroism; D006461:Hemolysis; D009154:Mutation; D010739:Phospholipase D / Q000737:chemistry* / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry; D013111:Spider Venoms / Q000737:chemistry / Q000201:enzymology*
chemical_list: D010743:Phospholipids; D013111:Spider Venoms; C055161:loxosceles venom; D010727:Phosphoric Diester Hydrolases; D010739:Phospholipase D
keywords: Activity modulation; Brown spider venom; Phospholipase D; Site-directed mutagenesis