Purification and characterization of a new L-amino acid oxidase from Daboia russellii siamensis venom.
A new L-amino acid oxidase (designated as DRS-LAAO) was purified from Daboia russellii siamensis venom by ion-exchange, gel filtration and affinity chromatographies. DRS-LAAO is a homodimeric enzyme with a molecular weight of 120.0 kDa as measured by size exclusion chromatography and the monomeric molecular weight of 58.0 kDa as measured by SDS-PAGE under both non-reducing and reducing conditions. The N-terminal amino acid sequence (ADDKNPLEECFREDD) of DRS-LAAO shares high identity with other snake venom L-amino acid oxidases, especially with those isolated from viperid venoms. The enzyme displayed high specificity towards hydrophobic L-amino acids. The best substrate of DRS-LAAO was L-Leu followed by L-Phe and L-Ile, while five substrates--L-Pro, L-Asn, L-Gly, L-Ser and L-Cys were not oxidized. Optimal pH of DRS-LAAO was 8.8. The enzyme showed no hemorrhagic activity even at a dosage of 55.0 microg. DRS-LAAO dose-dependently inhibited platelet aggregation induced by ADP (83.33 microM) and TMVA (55.0 nM) with an IC(50) value of 32.8 microg/ml and 32.3 microg/ml, respectively. The minimum inhibitory concentrations (MICs) of DRS-LAAO against Staphylococci aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922) were 9.0, 144.0 and 288.0 microg/ml, respectively. The minimum bactericidal concentrations (MBCs) of the enzyme for these strains were twice of the MIC values. These results showed that DRS-LAAO had the strongest antimicrobial activity against S. aureus among these three international standard stains. Antibacterial-activities of DRS-LAAO against eight clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates were also tested. The MICs of DRS-LAAO against these isolates ranged from 4.5 to 36.0 microg/ml. And the MBCs of the enzyme against these isolates ranged from 9.0 to 72.0 microg/ml.
Zhong
Shu-Rong
SR
Department of Animal Toxinology, Kunming Institute of Zoology, Chinese Academy of Sciences, 32 Jiaochang East Road, Kunming 650223, China.
Jin
Yang
Y
Wu
Jian-Bo
JB
Jia
Yong-Hong
YH
Xu
Gui-Li
GL
Wang
Gen-Chun
GC
Xiong
Yu-Liang
YL
Lu
Qiu-Min
QM
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects; D006801:Humans; D050830:L-Amino Acid Oxidase / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010974:Platelet Aggregation / Q000187:drug effects; D011550:Pseudomonas aeruginosa / Q000187:drug effects; D013211:Staphylococcus aureus / Q000187:drug effects; D013379:Substrate Specificity; D014757:Viper Venoms / Q000201:enzymology*
D014757:Viper Venoms; D050830:L-Amino Acid Oxidase