Oxidative transformation of tunichromes - Model studies with 1,2-dehydro-N-acetyldopamine and N-acetylcysteine.

Tunichromes are 1,2-dehydrodopa containing bioactive peptidyl derivatives found in blood cells of several tunicates. They have been implicated in metal sequestering, tunic formation, wound healing and defense reaction. Earlier studies conducted on these compounds indicate their extreme liability, high reactivity and easy oxidative polymerization. Their reactions are also complicated by the presence of multiple dehydrodopyl units. Since they have been invoked in crosslinking and covalent binding, to understand the reactivities of these novel compounds, we have taken a simple model compound that possess the tunichrome reactive group viz., 1,2-dehydro-N-acetyldopamine (Dehydro NADA) and examined its reaction with N-acetylcysteine in presence of oxygen under both enzymatic and nonenzymatic conditions. Ultraviolet and visible spectral studies of reaction mixtures containing dehydro NADA and N-acetylcysteine in different molar ratios indicated the production of side chain and ring adducts of N-acetylcysteine to dehydro NADA. Liquid chromatography and mass spectral studies supported this contention and confirmed the production of several different products. Mass spectral analysis of these products show the potentials of dehydro NADA to form side chain adducts that can lead to polymeric products. This is the first report demonstrating the ability of dehydro dopyl units to form adducts and crosslinks with amino acid side chains.

Oxidative transformation of a tunichrome model compound provides new insight into the crosslinking and defense reaction of tunichromes.

Tunichromes, small oligopeptides with dehydrodopa units isolated from the blood cells of ascidians, have been implicated in the defense reactions, metal binding, wound repair, or tunic formation. Their instability and high reactivity has severely hampered the assessment of their biological role. Experiments conducted with the model compound, 1,2-dehydro-N-acetyldopamine, indicated that the instability of tunichromes is due to this basic structure. Exposure of this catecholamine derivative to even mild alkaline condition such as pH 7.5 causes rapid nonenzymatic oxidation. High performance liquid chromatography and mass spectrometry studies confirmed the production of dimeric and other oligomeric products in the reaction mixture. The nonenzymatic reaction seemed to proceed through the intermediary formation of semiquinone free radical and superoxide anion. Ultraviolet and visible spectral studies associated with the oxidation of tunichromes isolated from Ascidia nigra by tyrosinase indicated the probable formation of oligomeric tunichrome products. Attempts to monitor the polymerization reaction by mass spectrometry ended in vain. Tunichrome also exhibited instability in mild alkaline conditions generating superoxide anions. Based on these studies, a possible role for oxidative transformation of tunichrome in defense reaction, tunic formation and wound healing is proposed.

Novel post-translational oligomerization of peptidyl dehydrodopa model compound, 1,2-dehydro-N-acetyldopa methyl ester.

Post-translational modification of peptidyl tyrosine to peptidyl dopa is widely observed in different marine organisms. While peptidyl dopas are oxidatively converted to dehydrodopa derivatives, nothing is known about the further fate of dehydrodopyl compounds. To fill this void, we studied the oxidation chemistry of a peptidyl dehydrodopa mimic, 1,2-dehydro-N-acetyldopa methyl ester with mushroom tyrosinase. We employed both routine biochemical studies and reversed phase liquid chromatography mass spectrometry to investigate the course of the reaction. Tyrosinase catalyzed the oxidation of 1,2-dehydro-N-acetyldopa methyl ester readily generating its typical o-quinone as the transient two-electron oxidation product. This quinone was extremely unstable and rapidly reacted with the parent compound forming benzodioxan type oligomeric products. Reaction mixture containing chemically made o-benzoquinone and 1,2-dehydro-N-acetyldopa methyl ester generated a mixed adduct of benzoquinone and 1,2-dehydro-N-acetyldopa methyl ester. Based on this finding, we propose that peptidyl dehydrodopa also exhibits a similar transformation accounting partially for the adhesive and cementing properties of dopyl proteins in nature.

Mass spectrometric studies shed light on unusual oxidative transformations of 1,2-dehydro-N-acetyldopa.

RATIONALE: Lamellarins are a group of over 70 plus bioactive marine natural compounds possessing a 6,7-dihydroxycoumarin moiety. Although they appear to derive from 3,4-dihydroxyphenylalanine (dopa), practically nothing is known about the metabolic fate of these compounds. Biochemical considerations indicate that they could arise from a N-acetyl-1,2-dehydrodopa precursor through oxidative cyclization reaction. METHODS: To assess the above hypothesis, we synthesized N-acetyl-1,2-dehydrodopa and conducted oxidation studies with commercially available mushroom tyrosinase and evaluated the course of the reaction with reversed-phase liquid chromatography/mass spectrometry (LC/MS). RESULTS: Mushroom tyrosinase readily oxidized N-acetyl-1,2-dehydrodopa - not to the normally expected quinone - but to an unstable quinone methide isomer, which rapidly cyclized to produce the dihydroxycoumarin product, 3-aminoacetyl esculetin. Interestingly, 3-aminoacetyl esculetin was further oxidized to a second quinone methide derivative that exhibited an addition reaction with the parent dihydroxycoumarin generating dimeric and other oligomeric products in the reaction mixture. CONCLUSIONS: LC/MS analysis of the N-acetyl-1,2-dehydrodopa oxidation reaction reveals not only a possible novel oxidative cyclization route for the biosynthesis of coumarin-type dehydrodopa compounds in marine organisms, but also unusual oxidative transformations of dehydro dopa derivatives.

On the mechanism of formation of arterenone in insect cuticular hydrolyzates.

Arterenone (2-amino-3',4'-dihydroxy acetophenone) is an important hydrolytic product generated from lightly colored sclerotized cuticle that use N-acyldopamine derivatives for crosslinking reactions. It seems to arise from 1,2-dehydro-N-acetyldopamine (dehydro NADA) that has been crosslinked to the cuticular components. However, the mechanism of generation of arterenone, which has two protons on the α-carbon and no proton on the ß-carbon atom from dehydro NADA crosslinks that have one proton each on these two side chain carbons, remained elusive and undetermined. To investigate the mechanism of this transformation, we synthesized specifically labeled ß-deuterated dehydro NADA and incubated with Sarcophaga bullata cuticle undergoing larval puparial transformation. We also isolated the dimeric products formed during the tyrosinase-mediated oxidation of dehydro NADA. Hydrolysis of both ß-deuterated dehydro NADA treated cuticle and ß-deuterated dehydro NADA dimer generated arterenone as the major hydrolytic product. Liquid chromatography-mass spectrometric analysis of this arterenone revealed the retention of deuterium from the ß-position of dehydro NADA at the α-carbon atom of arterenone. Hydrolysis of ß-deuterated dehydro NADA also generated the labeled arterenone under oxidative conditions, but not under anaerobic conditions. These results indicate the unique hydride shift from ß-carbon to α-carbon during acid hydrolysis and reveal the mechanism of liberation of arterenone and related compounds from dehydro NADA linked cuticle.

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.

Identification and quantification of histidine-rich glycoprotein (HRG) in the blood plasma of six marine bivalves.

Histidine-rich Glycoprotein (HRG) is a metal-binding protein described from the blood plasma of a pteriomorph bivalve, the marine mussel Mytilus edulis L. We demonstrate here, using Cd-Immobilized Metal Affinity Chromatography (IMAC), SDS-PAGE, Western Blotting, and ELISA, that HRG is present in three additional pteriomorphs and two heterodont bivalves. ELISA indicates that HRG is the predominant blood plasma protein in all six species (41 to 61% of total plasma proteins by weight). Thus, HRG appears to be a widespread metal-binding protein in the plasma of bivalves.

Genome sequence, comparative analysis and haplotype structure of the domestic dog.

Here we report a high-quality draft genome sequence of the domestic dog (Canis familiaris), together with a dense map of single nucleotide polymorphisms (SNPs) across breeds. The dog is of particular interest because it provides important evolutionary information and because existing breeds show great phenotypic diversity for morphological, physiological and behavioural traits. We use sequence comparison with the primate and rodent lineages to shed light on the structure and evolution of genomes and genes. Notably, the majority of the most highly conserved non-coding sequences in mammalian genomes are clustered near a small subset of genes with important roles in development. Analysis of SNPs reveals long-range haplotypes across the entire dog genome, and defines the nature of genetic diversity within and across breeds. The current SNP map now makes it possible for genome-wide association studies to identify genes responsible for diseases and traits, with important consequences for human and companion animal health.