Molecular approaches for structural characterization of Bothrops L-amino acid oxidases with antiprotozoal activity: cDNA cloning, comparative sequence analysis, and molecular modeling.
Two l-amino acid oxidases (LAAOs) were identified by random sequencing of cDNA libraries from the venom glands of Bothrops moojeni(BmooLAAO) and Bothrops jararacussu(Bjussu LAAO). Phylogenetic analysis involving other SV-LAAOs showed sequence identities within the range 83-87% being closely related to those from Agkistrodon and Trimeresurus. Molecular modeling experiments indicated the FAD-binding, substrate-binding, and helical domains of Bmoo and Bjussu LAAOs. The RMS deviations obtained by the superposition of those domains and that from Calloselasma rhodostoma LAAO crystal structure confirm the high degree of structural similarity between these enzymes. Purified BjussuLAAO-I and BmooLAAO-I exhibited antiprotozoal activities which were demonstrated to be hydrogen-peroxide mediated. This is the first report on the isolation and identification of cDNAs encoding LAAOs from Bothrops venom. The findings here reported contribute to the overall structural elucidation of SV-LAAOs and will advance the understanding on their mode of action.
França
Suzelei C
SC
Unidade de Biotecnologia Vegetal, Universidade de Ribeirão Preto, UNAERP, Ribeirão Preto-SP, Brazil. sfranca@unaerp.br
Kashima
Simone
S
Roberto
Patrícia G
PG
Marins
Mozart
M
Ticli
Fábio K
FK
Pereira
José Odair
JO
Astolfi-Filho
Spartaco
S
Stábeli
Rodrigo G
RG
Magro
Angelo J
AJ
Fontes
Marcos R M
MR
Sampaio
Suely V
SV
Soares
Andreimar M
AM
D000595:Amino Acid Sequence; D000818:Animals; D000981:Antiprotozoal Agents / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D017837:Bothrops; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D050830:L-Amino Acid Oxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
D000981:Antiprotozoal Agents; D017931:DNA Primers; D018076:DNA, Complementary; D050830:L-Amino Acid Oxidase