Rey-Suárez Paola, Acosta Cristian, Torres Uday, Saldarriaga-Córdoba Mónica, Lomonte Bruno, Núñez Vitelbina
Programa de Ofidismo y Escorpionismo, Facultad de Ciencias Farmacéuticas y Alimentarias, Universidad de Antioquia, Medellín, Colombia.
Centro de Investigación en Recursos Naturales y Sustentabilidad, Universidad Bernardo O'Higgins, Santiago de Chile, Chile.
PeerJ. 2018 Jun 8;6:e4924. doi: 10.7717/peerj.4924. eCollection 2018.
L-amino acid oxidases (LAAOs) are ubiquitous enzymes in nature. Bioactivities described for these enzymes include apoptosis induction, edema formation, induction or inhibition of platelet aggregation, as well as antiviral, antiparasite, and antibacterial actions. With over 80 species, snakes are the representatives of the Elapidae family in the New World. Although LAAOs in venoms have been predicted by venom gland transcriptomic studies and detected in proteomic studies, no enzymes of this kind have been previously purified from their venoms. Earlier proteomic studies revealed that the venom of from Colombia contains ∼4% of LAAO. This enzyme, here named MipLAAO, was isolated and biochemically and functionally characterized. The enzyme is found in monomeric form, with an isotope-averaged molecular mass of 59,100.6 Da, as determined by MALDI-TOF. Its oxidase activity shows substrate preference for hydrophobic amino acids, being optimal at pH 8.0. By nucleotide sequencing of venom gland cDNA of mRNA transcripts obtained from a single snake, six isoforms of MipLAAO with minor variations among them were retrieved. The deduced sequences present a mature chain of 483 amino acids, with a predicted pI of 8.9, and theoretical masses between 55,010.9 and 55,121.0 Da. The difference with experimentally observed mass is likely due to glycosylation, in agreement with the finding of three putative N-glycosylation sites in its amino acid sequence. A phylogenetic analysis of MmipLAAO placed this new enzyme within the clade of homologous proteins from elapid snakes, characterized by the conserved Serine at position 223, in contrast to LAAOs from viperids. MmipLAAO showed a potent bactericidal effect on (MIC: 2 µg/mL), but not on . The former activity could be of interest to future studies assessing its potential as antimicrobial agent.
L-氨基酸氧化酶（LAAOs）是自然界中普遍存在的酶。这些酶所具有的生物活性包括诱导细胞凋亡、形成水肿、诱导或抑制血小板聚集，以及抗病毒、抗寄生虫和抗菌作用。蛇类有80多个品种，是新大陆眼镜蛇科的代表。尽管通过毒腺转录组学研究预测了毒液中的LAAOs，并在蛋白质组学研究中检测到了它们，但此前尚未从其毒液中纯化出这类酶。早期的蛋白质组学研究表明，来自哥伦比亚的毒液中含有约4%的LAAO。这种酶在此被命名为MipLAAO，已被分离出来，并进行了生化和功能特性分析。通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）测定，该酶以单体形式存在，同位素平均分子量为59,100.6 Da。其氧化酶活性表现出对疏水氨基酸的底物偏好，在pH 8.0时活性最佳。通过对从一条蛇获得的mRNA转录本的毒腺cDNA进行核苷酸测序，检索到了六种MipLAAO同工型，它们之间存在微小差异。推导的序列显示有一条由483个氨基酸组成的成熟链，预测的等电点为8.9，理论质量在55,010.9至55,121.0 Da之间。与实验观察到的质量的差异可能是由于糖基化，这与在其氨基酸序列中发现的三个假定的N-糖基化位点一致。对MmipLAAO的系统发育分析将这种新酶置于眼镜蛇科同源蛋白的进化枝内，其特征是在第223位有保守的丝氨酸，这与蝰蛇科的LAAOs不同。MmipLAAO对[具体细菌名称1]显示出强大的杀菌作用（最低抑菌浓度：2 µg/mL），但对[具体细菌名称2]没有作用。前者的活性可能对未来评估其作为抗菌剂潜力的研究具有意义。
