The Chemistry of Escapin: Identification and Quantification of the Components in the Complex Mixture Generated by an L‐Amino Acid Oxidase in the Defensive Secretion of the Sea Snail Aplysia californica

Abstract

Escapin is an L-amino acid oxidase in the ink of a marine snail, the sea hare Aplysia californica, which oxidizes L-lysine (1) to produce a mixture of chemicals which is antipredatory and antimicrobial. The goal of our study was to determine the identity and relative abundance of the constituents of this mixture, using molecules generated enzymatically with escapin and also using products of organic syntheses. We examined this mixture under the natural range of pH values for ink-from approximately 5 at full strength to approximately 8 when fully diluted in sea water. The enzymatic reaction likely forms an equilibrium mixture containing the linear form alpha-keto-epsilon-aminocaproic acid (2), the cyclic imine Delta(1)-piperidine-2-carboxylic acid (3), the cyclic enamine Delta(2)-piperidine-2-carboxylic acid (4), possibly the linear enol 6-amino-2-hydroxy-hex-2-enoic acid (7), the alpha-dihydroxy acid 6-amino-2,2-dihydroxy-hexanoic acid (8), and the cyclic aminol 2-hydroxy-piperidine-2-carboxylic acid (9). Using NMR and mass spectroscopy, we show that 3 is the major component of this enzymatic product at any pH, but at more basic conditions, the equilibrium shifts to produce relatively more 4, and at acidic conditions, the equilibrium shifts to produce relatively more 2, 7, and/or 9. Studies of escapin's enzyme kinetics demonstrate that because of the high concentrations of escapin and L-lysine in the ink secretion, millimolar concentrations of 3, H(2)O(2), and ammonia are produced, and also lower concentrations of 2, 4, 7, and 9 as a result. We also show that reactions of this mixture with H(2)O(2) produce delta-aminovaleric acid (5) and delta-valerolactam (6), with 6 being the dominant component under the naturally acidic conditions of ink. Thus, the product of escapin's action on L-lysine contains an equilibrium mixture that is more complex than previously known for any L-amino acid oxidase.

Mechanisms of Action of Escapin, a Bactericidal Agent in the Ink Secretion of the Sea Hare Aplysia californica: Rapid and Long-Lasting DNA Condensation and Involvement of the OxyR-Regulated Oxidative Stress Pathway

“…Interestingly, the mixture of EIP-K plus H 2 O 2 , but not EIP-K, EEP-K, H 2 O 2 , or EEP-K plus H 2 O 2 , shows rapid, powerful, and longlasting bactericidal activity, much more than H 2 O 2 alone (22). The composition of EIP-K equilibrium mixture changes with the pH level (21). A cyclic deaminated product, ⌬ 1 -piperidine-2-carboxylic acid, dominates at any pH level, but the linear form, ␣-amino--caproic acid, becomes more prevalent at lower pH values (21), which is biologically significant because sea hare ink is acidic (27).…”

“…Escapin normally functions as an antipredatory chemical defense (12), but it also has broad antimicrobial activity, both bacteriostatic and bactericidal (34), indicative of its evolutionary roots (12,13). Escapin uses L-lysine as its primary substrate to produce diverse products (21,34). Escapin deaminates L-lysine to generate ␣-amino--caproic acid, H 2 O 2 , and ammonia (NH 3 ).…”

Defense through sensory inactivation: sea hare ink reduces sensory and motor responses of spiny lobsters to food odors

“…These mechanisms include phagomimicry, in which the chemical acts as a decoy (Kicklighter et al, 2005); chemical deterrency, in which the chemical is a repellent (Kicklighter et al, 2005;Aggio and Derby, 2008;Kamio et al, 2009;Kamio et al, 2010;Nusnbaum and Derby, 2010a;Nusnbaum and Derby, 2010b;Nusnbaum et al, 2012); and, as shown in this paper, sensory inactivation. These various forms of chemical defense in sea hares prove to be effective anti-predatory chemical defenses against diverse species under various conditions, such as hunger state of the predator and environmental availability of certain species of algae and thus diet-derived acquisition of deterrent compounds by the herbivorous sea hares (Derby, 2007;Derby and Aggio, 2011).…”

“…A second mechanism is as a repellent, as demonstrated against predatory fish, spiny lobsters, crabs and sea anemones (Nolen et al, 1995;Johnson and Willows, 1999;Kicklighter and Derby, 2006;Nusnbaum and Derby, 2010a). Several repellent molecules have been identified, including aplysioviolin, phycoerythrobilin and compounds in the 'escapin' pathway that include hydrogen peroxide, α-keto-ε-aminocaproic acid, Δ 1 -piperidine-2-carboxylic acid and others (Aggio and Derby, 2008;Nusnbaum and Derby, 2010a;Nusnbaum and Derby, 2010b;Kamio et al, 2009;Kamio et al, 2010). A candidate mechanism that has been proposed (Kicklighter et al, 2005;Derby, 2007) but not experimentally tested is sensory inactivation, in which the secretion decreases the activity of the predator's sensors such that the predator cannot detect appetitive stimuli.…”

Caracterização funcional e estudo dos mecanismos de resposta ao dano celular causado por uma L-aminoácido oxidase de Calloselasma rhodostoma em linhagens celulares humanas

“…As L-aminoácido oxidases (LAAOs, EC 1.4.3.2) são flavoenzimas encontradas em diferentes organismos, tais como bactérias, fungos, algas, peixes, caracóis e em peçonhas de serpentes das famílias Viperidae, Crotalidae e Elapidae (VALLON et al, 1993;CLEMETSON, 2002;KAMIO et al, 2009;CHEN et al, 2011;WANG et al, 2011;NUUTINEN et al, 2012, IZIDORO et al, 2014.…”