Abstract
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, Micrurus snakes are the representatives of the Elapidae family in the New World. Although LAAOs in Micrurus 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 M. mipartitus 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 S. aureus (MIC: 2 µg/mL), but not on E. coli. The former activity could be of interest to future studies assessing its potential as antimicrobial agent.
Highlights
L-amino acid oxidases (LAAOs, E.C. 1.4.3.2) are flavoenzymes that catalyze the stereospecific oxidative deamination of L-amino acid substrate to α-keto acid, producing ammonia and hydrogen peroxide (Izidoro et al, 2014)
The fraction identified as LAAO in M. mipartitus venom (Rey-Suárez et al, 2011), here named MipLAAO, was isolated using SEC-HPLC, with an retention time of 4.5 min, (Fig. 1A)
The cDNA obtained from the venom gland mRNA of M. mipartitus evidenced a product of ∼1,503 bp, corresponding to the expected molecular mass for the LAAO coding sequence
Summary
L-amino acid oxidases (LAAOs, E.C. 1.4.3.2) are flavoenzymes that catalyze the stereospecific oxidative deamination of L-amino acid substrate to α-keto acid, producing ammonia and hydrogen peroxide (Izidoro et al, 2014). In snake venoms (svLAAO) they are present in the Viperidae and Elapidae families, in amounts between 0.1 and 30% of total protein content (Izidoro et al, 2014) These enzymes have been found in non-venomous snakes such as Python regius and Pantherophis guttatus (Hargreaves et al, 2014). SvLAAOs are generally homodimeric glycoproteins (with approximately 4% of carbohydrates), with molecular masses ranging between 120 and 150 kDa in native forms, and from 55 to 66 kDa in monomeric forms, possibly with a non-covalent association between the two subunits (Du & Clemetson, 2002) They have a wide range of isoelectric points (pI), from about 4.4 to 8.5, and they can bind either to flavine mononucleotide (FMN) or to flavine adenine dinucleotide (FAD) (Izidoro et al, 2014). Most svLAAOs demonstrate a relatively high affinity for hydrophobic and aromatic amino acids, including L-Phe, L-Met, L-Leu and L-Ile because of substrate specificity related to side-chain binding sites (Costa et al, 2014; Geueke & Hummel, 2002), and they are sensitive to temperature, pH changes and lyophilization (Du & Clemetson, 2002)
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