Characterization of L-amino Acid Oxidase Derived from Crotalus adamanteus Venom: Procoagulant and Anticoagulant Activities

Abstract

Snake venom enzymes of the L-amino acid oxidase (LAAO) class are responsible for tissue hemorrhage, edema, and derangement of platelet function. However, what role, if any, these flavoenzymes play in altering plasmatic coagulation have not been well defined. Using coagulation kinetomic analyses (thrombelastograph-based), it was determined that the LAAO derived from Crotalus adamanteus venom displayed a procoagulant activity associated with weak clot strength (no factor XIII activation) similar to thrombin-like enzymes. The procoagulant activity was not modified in the presence of reduced glutathione, demonstrating that the procoagulant activity was likely due to deamination, and not hydrogen peroxide generation by the LAAO. Further, unlike the raw venom of the same species, the purified LAAO was not inhibited by carbon monoxide releasing molecule-2 (CORM-2). Lastly, exposure of the enzyme to phenylmethylsulfonyl fluoride (PMSF) resulted in the LAAO expressing anticoagulant activity, preventing contact activation generated thrombin from forming a clot. In sum, this investigation for the first time characterized the LAAO of a snake venom as both a fibrinogen polymerizing and an anticoagulant enzyme acting via oxidative deamination and not proteolysis as is the case with thrombin-like enzymes (e.g., serine proteases). Using this thrombelastographic approach, future investigation of purified enzymes can define their biochemical nature.

The Symmetric Active Site of Enantiospecific Enzymes

“…The high similarity of the overall three-dimensional fold and the very similar active site geometry support a strong evolutionary relationship among these enzymes [31]. The physiological role of LAAOs is often connected to their ability to generate hydrogen peroxide [48], a potent antimicrobial agent that can play an important role in microbial competition processes [49], biofilms dynamics [50], the protection of the fish skin from bacterial infections [51], and human immune system response [52,53]. Finally, LAAOs are also interesting for their potential biocatalytic applications [31].…”

“…The LAAO activity is widely distributed in nature, from bacteria [45,46] to mammals, which express LAAO in several tissues (e.g., in liver, kidney, brain, mammary gland, and polymorphonuclear leukocytes). In particular, snake venom represents the source of the best-characterized LAAOs [47,48]. Despite the low sequence identity between different LAAOs (e.g., the LAAO from the snake Crotalus adamanteus, CrLAAO, shares less than 23% sequence identity with the one from the bacterium Rhodococcus opacus), their overall fold is very similar, with a root mean squared deviation (RMSD) of ~1.0-1.2 Å when superimposed [31,47].…”

“…LAADs represent the most promising alternative to LAAOs for biotechnological applications since the latter enzyme cannot be efficiently expressed in a recombinant form. For example, LAADs have been proposed as biocatalysts for the production of optically pure D-amino acids through the resolution of D,L-racemic mixtures, as biological components in biosensors for the analytical determination of the concentration of L-amino acids [48,[71][72][73][74][75][76][77] and even as diagnostic or therapeutic agents [78][79][80].…”

Pulmonary involvement from animal toxins: the cellular mechanisms

“…The production of H 2 O 2 causes cytotoxicity to various cell types, including alveolar cells [ 77 ]. Additionally, LAAOs display both procoagulant activity by causing platelet aggregation and anticoagulant activity by weakening clots [ 79 ]. Intravenous injection of LAAO component in the pit viper Agkistrodon blomhoffii ussurensis venom to mice induced loss of pulmonary structure, pulmonary edema, and hemorrhage [ 77 ].…”

Direct Fluorescence Evaluation of d-Amino Acid Oxidase Activity Using a Synthetic d-Kynurenine Derivative

“…24 ■ EXPERIMENTAL SECTION Materials and Chemicals. Glutathione, 6-chloro-1,2benzisoxazol-3(2H)-one (CBIO), bovine serum albumin (BSA), xanthine oxidase from bovine milk (XOD), porcine kidney DAO (pkDAO; 6.4 U/mg), and L-amino acid oxidase from Crotalus adamanteus (LAO) 25 were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). Special-grade dimethyl sulfoxide (DMSO), 35% HCl, tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), acetic acid, boric acid, phosphoric acid, penicillin, streptomycin, sodium hydroxide, and LC-MS grade methanol (MeOH), 30% hydrogen peroxide (H 2 O 2 ), iron(II) sulfate (FeSO 4 ) heptahydrate, hypoxanthine (Hpx), sodium hypochlorite solution (available chlorine: 5.0%), potassium nitrite (KNO 2 ), uricase, and horseradish peroxidase (HRP) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).…”