Antibacterial action of a heat-stable form of L-amino acid oxidase isolated from king cobra (Ophiophagus hannah) venom Mui Li Lee a , Nget Hong Tan a, ⁎, Shin Yee Fung a , Shamala Devi Sekaran b a Department of Molecular Medicine, Faculty of Medicine, CENAR, University of Malaya, Kuala Lumpur, Malaysia b Department of Medical Microbiology, Faculty of Medicine, CENAR, University of Malaya, Kuala Lumpur, Malaysia abstract article info Article history: Received 14 September 2010 Received in revised form 1 November 2010 Accepted 1 November 2010 Available online 6 November 2010 Keywords: Ophiophagus hannah venom L-amino acid oxidase Antibacterial action The major L-amino acid oxidase (LAAO, EC 1.4.3.2) of king cobra (Ophiophagus hannah) venom is known to be an unusual form of snake venom LAAO as it possesses unique structural features and unusual thermal stability. The antibacterial effects of king cobra venom LAAO were tested against several strains of clinical isolates including Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli using broth microdilution assay. For comparison, the antibacterial effects of several antibiotics (cefotaxime, kanamycin, tetracycline, vancomycin and penicillin) were also examined using the same conditions. King cobra venom LAAO was very effective in inhibiting the two Gram-positive bacteria (S. aureus and S. epidermidis) tested, with minimum inhibitory concentration (MIC) of 0.78 μg/mL (0.006 μM) and 1.56 μg/mL (0.012 μM) against S. aureus and S. epidermidis, respectively. The MICs are comparable to the MICs of the antibiotics tested, on a weight basis. However, the LAAO was only moderately effective against three Gram-negative bacteria tested (P. aeruginosa, K. pneumoniae and E. coli), with MIC ranges from 25 to 50 μg/mL (0.2–0.4 μM). Catalase at the concentration of 1 mg/mL abolished the antibacterial effect of LAAO, indicating that the antibacterial effect of the enzyme involves generation of hydrogen peroxide. Binding studies indicated that king cobra venom LAAO binds strongly to the Gram-positive S. aureus and S. epidermidis, but less strongly to the Gram-negative E. coli and P. aeruginosa, indicating that specific binding to bacteria is important for the potent antibacterial activity of the enzyme. © 2010 Elsevier Inc. All rights reserved. 1. Introduction L-amino acid oxidase (L-amino acid: O 2 oxidoreductase, EC 1.4.3.2, abbreviation LAAO) is a dimeric flavoenzyme containing non- covalently bound FAD or FMN as a prosthetic group. It catalyzes the oxidative deamination of an L-amino acid to produce the corresponding α-keto acid, hydrogen peroxide and ammonia via imino acid intermediate. Snake venoms are rich sources of LAAO (Du and Clemetson, 2002; Tan and Fung, 2009). The enzyme exhibits a wide range of biological activities including apoptosis-inducing, edema-inducing, inhibition or induction of platelet aggregation, antibacterial effect and antiviral activity (Tan and Fung, 2009). The role of LAAO in the pharmacological action of snake venom, however, is still not fully understood. Generally, the enzyme has a low lethal toxicity in mice. Skarnes (1970) was the first to report the bactericidal activity of a LAAO isolated from Crotalus adamanteus venom. In the last 20 years, many authors have also reported the antibacterial activity of LAAOs from snake venoms (see Table 2 for a list) and other animals such as giant African snail and sea hares (Ehara et al., 2002; Yang et al., 2005; Nagashima et al., 2009). Antibacterial action of LAAOs appears to result from hydrogen peroxide generated by the oxidative action of the enzyme, as the effect is abolished in the presence of hydrogen peroxide scavengers such as catalase (Tan and Fung, 2009). Several authors have reported isolation and characterization of L- amino acid oxidase from king cobra (Ophiophagus hannah) venom (Tan and Saifuddin, 1989, 1991; Li et al., 1994; Ahn et al., 1997; Jin et al., 2007). Tan and Saifuddin (1989) reported that king cobra venom LAAO exhibited unusual thermal stability. At pH 7.4, the enzyme retained 100% activity after incubation at 25 °C for 30 days. They also reported that unlike other snake venom LAAO, king cobra venom LAAO was stable at alkaline condition and was not inactivated by freezing. The enzyme also exhibited unique substrate specificity: it was very active against L-lysine, which was a poor substrate for other snake venom LAAOs. Structural studies showed that indeed king cobra venom LAAO is evolutionarily distant to other snake venom LAAOs (Jin et al., 2007). The substrate specificity and thermal stability of the enzyme are, however, similar to LAAO isolated from marine sources such as sea hare (Yang et al., 2005). In view of the unusual thermal stability and unique structural feature and substrate specificity of the LAAO, it would be interesting to investigate the antibacterial action of Comparative Biochemistry and Physiology, Part C 153 (2011) 237–242 ⁎ Corresponding author. Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia. Tel.: + 60 3 79674912; fax: + 60 3 79674957. E-mail address: tanngethong@yahoo.com.sg (N.H. Tan). 1532-0456/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2010.11.001 Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part C journal homepage: www.elsevier.com/locate/cbpc