Reexamination of the mechanisms of oxidative transformation of the insect cuticular sclerotizing precursor, 1,2-dehydro-N-acetyldopamine

1,2-dehydro-N-acetyldopamine (dehydro NADA) is an important catecholamine derivative formed during the sclerotization of insect cuticle. Earlier we have reported that tyrosinase-catalyzed oxidation of dehydro NADA produces a reactive quinone methide imine amide that forms adducts and cross-links through its side chain, thereby accounting for sclerotization reactions. Recently, laccase has also been identified as a key enzyme associated with sclerotization. Hence, we re-examined oxidation of dehydro NADA by tyrosinase and laccase using high performance liquid chromatography tandem mass spectrometry. Tyrosinase-catalyzed oxidation of dehydro NADA not only generated dimers as reported earlier, but also generated significant amounts of oligomers. The course of laccase-catalyzed oxidation of dehydro NADA significantly differed from the tyrosinase reaction kinetically and mechanistically. Laccase failed to produce any detectable quinone or quinone methide as the primary two-electron oxidation product. Since laccases are known to generate primarily semiquinones as the initial products, lack of accumulation of two-electron oxidation products indicated that laccase reaction is primarily occurring via free radical coupling mechanism. Consistent with this proposal, laccase-catalyzed oxidation of dehydro NADA, resulted in the production of largely dimeric products and failed to produce any significant amount of oligomeric materials. These studies call for radical coupling as yet another major mechanism for sclerotization of insect cuticle. (C) 2010 Elsevier Ltd. All rights reserved.