Cloning, characterization and mutagenesis of Russell's viper venom L-amino acid oxidase: Insights into its catalytic mechanism
Resource
BIOCHIMIE, 94(2), 335-344
Journal
Biochimie
Pages
335-344
Date Issued
2012
Date
2012
Author(s)
Chen, Hong-Sen
Wang, Ying-Ming
Huang, Wan-Ting
Huang, Kai-Fa
Tsai, Inn-Ho
Abstract
To investigate the structure-function relationships and geographic variations of L-amino acid oxidase (LAAO) from Daboia venoms, a single LAAO (designated as DrLAO) was purified from eastern Indian Daboia russelii venom and characterized. The purified DrLAO showed subunit molecular mass of 60-64kDa; its N-terminal sequence (1-20) was identical to those of several true viper LAAOs. Its preferred substrates were hydrophobic l-amino acids and the kinetic specificities were ordered as follows: Phe, Tyr, Met, Leu, and Trp. Enzyme assay and Western blotting showed that the venom LAAO contents of D. russelii were higher than those of Daboia siamensis. DrLAO dose-dependently inhibited ADP- and collagen-induced platelet aggregation with IC(50) values of 0.27 and 0.82μM, respectively. Apparently, DrLAO may synergize with other venom components to prolong and enhance bleeding symptoms after Daboia envenoming. The full sequence of DrLAO was deduced from its cDNA sequence and then confirmed by peptide mass fingerprinting. Molecular phylogenetic analysis revealed that SV-LAAO family members could be differentiated not only by snake taxonomy but also by the variations at position 223, and they divided into H223, S223, N223, and D223 subclasses. We have further prepared recombinant DrLAO and mutants by the Pichia expression system. Mutagenic analyses of DrLAO His223 revealed that this residue bound substrates instead of serving as an essential base in the catalytic steps. Our results suggest a direct hydride transfer from substrate to FAD as the mechanism for SV-LAAOs.
SDGs
Type
journal article
File(s)![Thumbnail Image]()
Loading...
Name
31.pdf
Size
23.23 KB
Format
Adobe PDF
Checksum
(MD5):b0e05ee15d2bd5e37733e960867774e7