Unraveling complex molecular transformations of N-ß-alanyldopamine that account for brown coloration of insect cuticle.

RATIONALE: N-ß-Alanyldopamine (NBAD) and N-acetyldopamine (NADA) are catecholamines that are used by insects as sclerotizing precursors to harden their cuticle. They share a common pathway utilizing the same set of sclerotizing enzymes. Yet, cuticles using NBAD are brown, while cuticles using NADA are colorless. To identify the cause of this major unresolved color difference, molecular transformations of NBAD with cuticular enzymes were investigated. METHODS: Reactions of NBAD and NADA with native cuticle isolated from the wandering stages of Sarcophaga bullata larvae as well as the reactions of NBAD with cuticular sclerotization enzymes - phenoloxidase, quinone isomerase and quinone methide isomerase - were investigated using UV-Vis spectroscopy, high-performance liquid chromatography (HPLC), and mass spectrometry (MS). In addition, the reactivity of enzymatically generated NBAD quinone was investigated by MS. RESULTS: Reactions of NBAD with sclerotizing enzymes isolated from Sarcophaga bullata larvae generate colorless products such as N-ß-alanylnorepinephrine, N-ß-alanylarterenone, dehydro NBAD, the benzodioxan dimers of dehydro NBAD and other minor adducts, the same kind of compounds generated by NADA reaction with cuticular enzymes. However, oxidation of NBAD produces colored quinone adducts, in addition. NADA, which lacks the amino group, did not produce these quinone adducts. CONCLUSIONS: LC/MS analysis of the reaction mixture of NBAD-cuticular enzyme reactions reveals the novel production of colored quinone adducts that are not possible for NADA. Therefore, our results suggest that the brown coloration of cuticle formed through NBAD crosslinking is likely due to the formation and accumulation of NBAD quinone and its adducts, while NADA quinone adducts tend not to form during NADA crosslinking, producing a nearly colorless cuticle.

Critical Analysis of the Melanogenic Pathway in Insects and Higher Animals.

Animals synthesize melanin pigments for the coloration of their skin and use it for their protection from harmful solar radiation. Insects use melanins even more ingeniously than mammals and employ them for exoskeletal pigmentation, cuticular hardening, wound healing and innate immune responses. In this review, we discuss the biochemistry of melanogenesis process occurring in higher animals and insects. A special attention is given to number of aspects that are not previously brought to light: (1) the molecular mechanism of dopachrome conversion that leads to the production of two different dihydroxyindoles; (2) the role of catecholamine derivatives other than dopa in melanin production in animals; (3) the critical parts played by various biosynthetic enzymes associated with insect melanogenesis; and (4) the presence of a number of important gaps in both melanogenic and sclerotinogenic pathways. Additionally, importance of the melanogenic process in insect physiology especially in the sclerotization of their exoskeleton, wound healing reactions and innate immune responses is highlighted. The comparative biochemistry of melanization with sclerotization is also discussed.

Drosophila yellow-h encodes dopaminechrome tautomerase: A new enzyme in the eumelanin biosynthetic pathway.

Melanin is a widely distributed phenolic pigment that is biosynthesized from tyrosine and its hydroxylated product, dopa, in all animals. However, recent studies reveal a significant deviation from this paradigm, as insects appear to use dopamine rather than dopa as the major precursor of melanin. This observation calls for a reconsideration of the insect melanogenic pathway. While phenoloxidases and laccases can oxidize dopamine for dopaminechrome production, the fate of dopaminechrome remains undetermined. Dopachrome decarboxylase/tautomerase, encoded by yellow-f/f2 of Drosophila melanogaster, can convert dopaminechrome into 5,6-dihydroxyindole, but the same enzyme from other organisms does not act on dopaminechrome, suggesting the existence of a specific dopaminechrome tautomerase (DPT). We now report the identification of this novel enzyme that biosynthesizes 5,6-dihydroxyindole from dopaminechrome in Drosophila. Dopaminechrome tautomerase acted on both dopaminechrome and N-methyl dopaminechrome but not on dopachrome or other aminochromes tested. Our biochemical and molecular studies reveal that this enzyme is encoded by the yellow-h gene, a member of the yellow gene family, and advance our understanding of the physiological functions of this gene family. Identification and characterization of DPT clarifies the precursor for melanin biosynthetic pathways and proves the existence of an independent melanogenic pathway in insects that utilizes dopamine as the primary precursor.

Drosophila melanogaster has the enzymatic machinery to make the melanic component of neuromelanin.

In Drosophila, the same set of genes that are used for cuticle pigmentation and sclerotization are present in the nervous system and are responsible for neurotransmitter recycling. In this study, we carried out biochemical analysis to determine whether insects have the enzymatic machinery to make melanic component of neuromelanin. We focused our attention on two key enzymes of melanogenesis, namely phenoloxidase and dopachrome decarboxylase/tautomerase. Activity staining of the proteins isolated from the Drosophila larval brain tissue, separated by native polyacrylamide gel electrophoresis, indicated the presence of these two enzymes. Mass spectral sequence analysis of the band also supported this finding. To best of our knowledge, this is the first report on the presence of the enzymatic machinery to make melanin part of neuromelanin in any insect brain.

Insect cuticular melanins are distinctly different from those of mammalian epidermal melanins.

Melanin from several insect samples was isolated and subjected to chemical degradation and HPLC analysis for melanin markers. Quantification of different melanin markers reveals that insect melanins are significantly different from that of the mammalian epidermal melanins. The eumelanin produced in mammals is derived from the oxidative polymerization of both 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acids. The pheomelanin is formed by the oxidative polymerization of cysteinyldopa. Thus, dopa is the major precursor for both eumelanin and pheomelanin in mammals. But insect eumelanin appears to be mostly made from 5,6-dihydroxyindole and originates from dopamine. More importantly, our study points out the wide spread occurrence of pheomelanin in many insect species. In addition, cysteinyldopamine and not cysteinyldopa is the major precursor for insect pheomelanin. Thus, both eumelanin and pheomelanin in insects differ from higher animals using dopamine and not dopa as the major precursor.