Biochemical, functional and structural characterization of Akbu-LAAO: a novel snake venom L-amino acid oxidase from Agkistrodon blomhoffii ussurensis.
An L-amino acid oxidase (Akbu-LAAO) was isolated from the venom of Agkistrodon blomhoffii ussurensis snake using DEAE Sephadex A-50 ion-exchange, Sephadex G-75 gel filtration, and high performance liquid chromatographies. The homogeneity and molecular mass of Akbu-LAAO were analyzed by SDS-PAGE and MALDI-TOF spectrometry. The sequences of ten peptides from Akbu-LAAO were established by HPLC-nESI-MS/MS analysis. Protein sequence alignment indicated that i) that Akbu-LAAO is a new snake venom LAAO, and ii) Akbu-LAAO shares homology with several LAAOs from the venoms of Calloselasma rhodost, Agkistrodon halys, Daboia russellii siamensis, and Trimeresurus stejnegeri. Akbu-LAAO is a homodimer with a molecular mass of approximately 124.4 kDa. It reacts optimally with its enzymatic substrate, Leu, at pH 4.7 with a K(m) of 2.1 mM. ICP-AES measurements showed that Akbu-LAAO contains four Zn(2+) per dimer that are unessential for the hydrolytic activity of the enzyme. The emission fluorescence intensity of Akbu-LAAO decreases by 61% on removal of Zn(2+) indicating that the zinc probably helps maintain the structural integrity of the enzyme. The addition of exogenous metal ions, including Mg(2+), Mn(2+), Ca(2+), Ce(3+), Nd(3+), Co(2+) and Tb(3+), increases the l-Leu hydrolytic activity of the enzyme. Akbu-LAAO shows apparent anti-aggregation effects on human and rabbit platelets. It exhibits a strong bacteriostasis effect on Staphylococcus aureus, eighteen fold that of cephalosporin C under the same conditions. Taken together, the biochemical, proteomic, structural and functional characterizations reveal that Akbu-LAAO is a novel LAAO with promise for biotechnological and medical applications.
Sun
Ming-Zhong
MZ
Department of Biotechnology, Dalian Medical University, Dalian, China.
Guo
Chunmei
C
Tian
Yuxiang
Y
Chen
Duo
D
Greenaway
Frederick T
FT
Liu
Shuqing
S
D017836:Agkistrodon; D000595:Amino Acid Sequence; D000818:Animals; D003435:Crotalid Venoms / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D050830:L-Amino Acid Oxidase / Q000302:isolation & purification* / Q000378:metabolism; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D010974:Platelet Aggregation / Q000187:drug effects; D055503:Protein Multimerization; D011817:Rabbits; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013054:Spectrophotometry, Atomic; D013211:Staphylococcus aureus / Q000187:drug effects; D015032:Zinc / Q000737:chemistry
D003435:Crotalid Venoms; D008670:Metals; D050830:L-Amino Acid Oxidase; D015032:Zinc