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.     

Biosynthesis of dehydro-N-acetyldopamine by a soluble enzyme preparation from the larval cuticle of Sarcophaga bullata involves intermediary formation of N-acetyldopamine quinone and N-acetyldopamine quinone methide

The enzymes involved in the side chain hydroxylation and side chain desaturation of the sclerotizing precursor N-acetyldopamine (NADA) were obtained in the soluble form from the larval cuticle of Sarcophaga bullata and the mechanism of the reaction was investigated. Phenylthiourea, a well-known inhibitor of phenoloxidases, drastically inhibited both the reactions, indicating the requirement of a phenoloxidase component. N-acetylcysteine, a powerful quinone trap, trapped the transiently formed NADA quinone and prevented the production of both N-acetylnorepinephrine and dehydro NADA. Exogenously added NADA quinone was readily converted by these enzyme preparations to N-acetylnorepinephrine and dehydro NADA. 4-Alkyl-o-quinone:2-hydroxy-p-quinone methide isomerase obtained from the cuticular preparations converted chemically synthesized NADA quinone to its quinone methide. The quinone methide formed reacted rapidly and nonenzymatically with water to form N-acetylnorepinephrine as the stable product. Similarly 4-(2-hydroxyethyl)-o-benzoquinone was converted to 3,4-dihydroxyphenyl glycol. When the NADA quinone-quinone isomerase reaction was performed in buffer containing 10% methanol, beta-methoxy NADA was obtained as an additional product. Furthermore, the quinones of N-acetylnorepinephrine and 3,4-dihydroxyphenyl glycol were converted to N-acetylarterenone and 2-hydroxy-3',4'-dihydroxyacetophenone, respectively, by the enzyme. Comparison of nonenzymatic versus enzymatic transformation of NADA to N-acetylnorepinephrine revealed that the enzymatic reaction is at least 100 times faster than the nonenzymatic rate. Resolution of the NADA desaturase system on Benzamidine Sepharose and Sephacryl S-200 columns yielded the above-mentioned quinone isomerase and NADA quinone methide:dehydro NADA isomerase. The latter, on reconstitution with mushroom tyrosinase and hemolymph quinone isomerase, catalyzed the biosynthesis of dehydro NADA from NADA with the intermediary formation of NADA quinone and NADA quinone methide. The results are interpreted in terms of the quinone methide model elaborated by our group [Sugumaran: Adv. Insect Physiol. 21:179-231, 1988; Sugumaran et al.: Arch. Insect Biochem. Physiol. 11:109, 1989] and it is concluded that the two enzyme beta-sclerotization model [Andersen: Insect Biochem. 19:59-67, 375-382, 1989] is inadequate to account for various observations made on insect cuticle.     

Mechanism of activation of 1,2-dehydro-N-acetyldopamine for cuticular sclerotization

The mechanism of oxidation of 1,2-dehydro-N-acetyldopamine (dehydro NADA) was examined to resolve the controversy between our group and Andersen's group regarding the reactive species involved in β-sclerotization. While Andersen has indicated that dehydro NADA quinone is the β-sclerotizing agent [Andersen, 1989], we have proposed quinone methides as the reactive species for this process [Sugumaran, 1987; Sugumaran, 1988]. Since dehydro NADA quinone has not been isolated or identified till to date, we studied the enzymatic oxidation of dehydro NADA in the presence of quinone traps to characterize this intermediate. Accordingly, both N-acetylcysteine and o-phenylenediamine readily trapped the transiently formed dehydro NADA quinone as quinone adducts. Interestingly, when the enzymatic oxidation was performed in the presence of o-aminophenol or different catechols, adduct formation between the dehydro NADA side chain and the additives had occurred. The structure of the adducts is in conformity with the generation and reactions of dehydro NADA quinone methide (or its radical). This, coupled with the fact that 4-hydroxyl or amino-substituted quinones instantly transformed into p-quinonoid structure, indicates that dehydro NADA quinone is only a transient intermediate and that it is the dehydro NADA quinone methide that is the thermodynamically stable product. However, since this compound is chemically more reactive due to the presence of both quinone methide and acylimine structure on it, the two side chain carbon atoms are “activated.” Based on these considerations, it is suggested that the quinone methide derived from dehydro NADA is the reactive species responsible for cross-link formation between dehydro NADA and cuticular components during β-sclerotization.     

Catechols involved in sclerotization of cuticle and egg pods of the grasshopper,Melanoplus sanguinipes,and their interactions with cuticular proteins

N‐Acetyldopamine (NADA) is the major catechol in the hemolymph of nymphal and adult grasshoppers, Melanoplus sanguinipes (F.), and mainly occurs as an acid‐labile conjugate indicated to be a sulfate ester. Its concentration increases in last instar nymphs and peaks during adult cuticle sclerotization. Dopamine (DA), the precursor of NADA and melanic pigments, is about 10 times lower in concentration than NADA, but shows a similar pattern of accumulation. NADA also predominates in cuticle, but its concentration is lowest during the active period of sclerotization, reflecting its role as a precursor for quinonoid tanning agents. Two other catechols, 3,4‐dihydroxybenzoic acid (DOBA) and 3,4‐dihydroxyphenylethanol (DOPET), also occur in hemolymph and cuticle, and their profiles suggest a role in cuticle stabilization. Solid‐state NMR analysis of sclerotized grasshopper cuticle (fifth instar exuviae) estimated the relative abundances of organic components to be 59% protein, 33% chitin, 6% catechols, and 2% lipid. About 99% of the catechols are covalently bound in the cuticle, and therefore are involved in sclerotization of the protein‐chitin matrix. To determine the types of catechol covalent interactions in the exocuticle, samples of powdered exuviae were heated in Hcl under different hydrolytic conditions to release adducts and cross‐linked products. 3,4‐Dihydroxyphenylketoethanol (DOPKET) and 3,4‐dihydroxyphenylketoethylamine (arterenone) are the major hydrolysis products in weak and strong acid, respectively, and primarily represent NADA oligomers that apparently serve as cross‐links and filler material in sclerotized cuticle. Intermediate amounts of norepinephrine (NE) are released, which represent N‐acetylnorepinephrine (NANE), a hydrolysis product of NADA bonded by the b‐carbon to cuticular proteins and possibly chitin. Small quantities of histidyl‐DA and histidyl‐DOPET ring and side‐chain C‐N adducts are released by strong acid hydrolysis. Therefore, grasshopper cuticle appears to be sclerotized by both o‐quinones and p‐quinone methides of NADA and dehydro‐NADA, which results in a variety of C‐O and C‐N covalent bonds linked primarily through the side‐chain carbons of the catechol moiety to amino acid residues in cuticular proteins. The primary catechol extracted from both the female accessory glands/calyx and the proteinaceous frothy material of the egg pod is DOBA, which also commonly occurs in cockroach accessory glands and oothecae, presumably as a tanning agent precursor. 3,4‐Dihydroxyphenylalanine (DOPA) was also detected in extracts of the accessory glands/calyx of grasshoppers, and may serve as a precursor for DOBA synthesis. Arch. Insect Biochem. Physiol. 40:119–128, 1999. © 1999 Wiley‐Liss, Inc.     

Further studies on the mechanism of oxidation of N-acetyldopamine by the cuticular enzymes from Sarcophaga bullata and other insects

In accordance with our earlier results, quinone methide formation was confirmed to be the major pathway for the oxidation of N-acetyldopamine (NADA) by cuticle-bound enzymes from Sarcophaga bullata larvae. In addition, with the use of a newly developed HPLC separation condition and cuticle prepared by gentle procedures, it could be demonstrated that 1, 2-dehydro-NADA and its dimeric oxidation products are also generated in the reaction mixture containing a high concentration of NADA albeit at a much lower amount than the NADA quinone methide water adduct, viz., N-acetylnorepinephrine (NANE). By using different buffers, it was also possible to establish the accumulation of NADA quinone in reaction mixtures containing NADA and cuticle. That the 1,2-dehydro-NADA formation is due to the action of a NADA desaturase system was established by pH and temperature studies and by differential inhibition of NANE production. Of the various cuticle examined, adult cuticle of Locusta migratoria, presclerotized cuticle of Periplaneta americana, and white puparial cases of Drosophila melanogaster exhibited more NADA desaturase activity than NANE generating activity, while the reverse was observed with the larval cuticle of Tenebrio molitor and pharate pupal cuticle of Manduca sexta. These studies indicate that both NADA quinone methide and 1, 2-dehydro NADA are formed during enzymatic activation of NADA in insect cuticle. Based on these results, a unified mechanism for β-sclerotization involving quinone methides as the reactive species is presented.     

Enzymatic activities involved in incorporation of N-acetyldopamine into insect cuticle during sclerotization

During sclerotization of insect cuticle, N-acetyldopamine (NADA) is enzymatically oxidized before reaction with cuticular proteins. Not all oxidized NADA reacts with cuticular structural materials, a small fraction reacts with water or other available low molecular weight compounds to give soluble products. Various types of cuticle were incubated with excess NADA and the products studied by reversed phase high performance liquid chromatography (RP-HPLC) to obtain information on the enzymatic activities in the cuticle. The occurrence of at least two enzymes competing for NADA and present in different proportions in the various types of cuticle can explain the results. NADA may be incorporated into cuticle via α,β-dehydro-NADA (β-sclerotization) or via quinone methides and o-quinones, and the actual course of sclerotization will depend upon the relative activities of the enzymes involved. The various pathways may all be used simultaneously.     

Investigation of an Ortho-quinone isomerase from larval cuticle of the American silkmoth,Hyalophora cecropia

Insect cuticles catalyze the formation of N-acetylnorepinephrine (NANE) and N-β-alanylnorepinephrine (NBANE) from N-acetyldopamine (NADA) and N-β-alanyldopamine (NBAD), respectively. An enzyme, involved in the reaction, has now been isolated from fifth stage larval cuticle of Hyalophora cecropia and partially characterized. The enzyme alone has hardly any activity towards NADA, but together with diphenoloxidases [catechol oxidases (EC 1.10.3.1) or laccases (EC 1.10.3.2)] it will produce NANE as the main product from NADA, indicating that NADA-quinone is the actual substrate for the enzyme. The enzyme is presumably an ortho-quinone para-quinone methide isomerase, and formation of NANE is due to non-enzymatic addition of water to the quinone methide. The enzyme combination mushroom tyrosinase-cuticular isomerase has pH optimum at 5.5, and the optimal substrate concentration is about 10 mM NADA.Together with the endogenous cuticular diphenoloxidases the isomerase can account for the formation of NANE observed when pieces of intact cuticle are incubated with NADA, and for the presence of NANE and NBANE in sclerotized cuticle.The possible roles of the enzyme in sclerotization and defense reactions in insects are briefly discussed.     

The effects of pH on the rates of isotope exchange catalyzed by alanine aminotransferase.

Alanine aminotransferase catalyzes exchange of the β-hydrogens of alanine with the solvent at a rate commensurate with the rate of exchange of the α-hydrogen. These methyl protons are lost sequentially and intermediates having protons on the α-carbon but deuterium on the β-carbon were detected by nuclear magnetic resonance. The overall rates of exchange of both α-hydrogen and β-hydrogen were less than the rate of transamination and did not vary from pH 6–8. The α-hydrogen of glutamate, on the other hand, was found to exchange at a greater rate than the overall transamination rate with ketoglutarate. However the β-hydrogens of glutamate are not removed during the enzymic reaction. It is concluded that a basic group on the enzyme removes the proton from the α-carbon of alanine at a rate at least as great as the rate of transamination. Because the proton is held on the enzyme, it appears to exchange more slowly in alanine. Labilization of the α-hydrogen of amino acids does not appear to be the ratelimiting reaction of alanine aminotransferase, but occurs at a rate comparable to that of the overall reaction.     

Differential mechanism of oxidation of N-acetyldopamine and N-acetylnorepinephrine by cuticular phenoloxidase from Sarcophaga bullata

The mechanism of oxidation of two related sclerotizing precursors—N-acetyldopamine and N-acetylnorepinephrine—by the cuticular phenoloxidase from Sarcophaga bullata was studied and compared with mushroom tyrosinase-mediated oxidation. While the fungal enzyme readily generated the quinone products from both of these catecholamine derivatives, sarcophagid enzyme converted N-acetyldopamine to a quinone methide derivative, which was subsequently bound to the cuticle with the regeneration of o-dihydroxy phenolic function as outlined in an earlier publication [Sugumaran: Arch Insect Biochem Physiol, 8, 73 (1988)]. However, it converted N-acetylnorepinephrine to its quinone and not to the quinone methide derivative. Proteolytic digests of N-acetyldopamine-treated cuticle liberated peptides that had covalently bound catechols, while N-acetylnorepinephrine-treated cuticle did not release such peptides. Acid hydrolysis of N-acetyldopamine-treated cuticle, but not N-acetylnorepinephrine-treated cuticle liberated 2-hydroxy-3′,4′-dihydroxyacetophenone and arterenone. These results further confirm the unique conversion of N-acetyldopamine to its corresponding quinone methide derivative and N-acetylnorepinephrine to its quinone derivative by the cuticular phen-oloxidase. Significance of this differential mechanism of oxidation for sclerotization of insect cuticle is discussed.     

A nuclear magnetic resonance study of the helix–coil transition of poly(L-glutamic acid)

There have been many reports that the nuclear magnetic resonance (nmr) spectra of a large number of polypeptides exhibit peak doubling of the α-carbon and the α-carbon proton in the helix–coil transition region. One apparent exception to this generalization has been polypeptides with ionizable side chains, where the helix–coil transition is induced by changes in pH in aqueous solution. Because it is important to establish the proper theoretical reason for the peak doubling and its relation to the rate of conformational change of amino acid residues, we have reexamined the proton and carbon-13 nmr spectra, at high field, for two polydisperse samples of poly(L -glutamic acid). Doubling of the α-carbon proton resonance as well as those of the α- and β-carbon, and backbone carbonyl are observed for a low-molecular-weight sample (DP = 54), while a higher molecular weight sample (DP = 309), exhibits only single resonances. Thus, polydispersity by itself is not sufficient to observe peak doubling; low-molecular weight is also required.     

Aspects of cuticular sclerotization in the locust, Scistocerca gregaria, and the beetle, Tenebrio molitor

The number of reactive amino groups in cuticular proteins decreases during the early period of insect cuticular sclerotization, presumably due to reaction with oxidation products of N-acetyldopamine (NADA) and N-beta-alanyldopamine (NBAD). We have quantitated the decrease in cuticular N-terminal amino groups and lysine epsilon-amino groups during the first 24h of sclerotization in adult locusts, Schistocerca gregaria, and in larval and adult beetles, Tenebrio molitor, as well as the increase in beta-alanine amino groups in Tenebrio cuticle. The results indicate that nearly all glycine N-terminal groups and a significant part of the epsilon-amino groups from lysine residues are involved in the sclerotization process in both locusts and Tenebrio. A pronounced increase in the amount of free beta-alanine amino groups was observed in cuticle from adult Tenebrio and to a lesser extent also in Tenebrio larval cuticle, but from locust cuticle no beta-alanine was obtained. Hydrolysis of sclerotized cuticles from locusts and Tenebrio by dilute hydrochloric acid released a large number of compounds containing amino acids linked to catecholic moieties. Products have been identified which contain histidine residues linked via their imidazole group to the beta-position of various catechols, such as dopamine, 3,4-dihydroxyphenyl-ethanol (DOPET), and 3,4-dihydroxyphenyl-acetaldehyde (DOPALD), and a ketocatecholic compound has also been identified composed of lysine linked via its epsilon-amino group to the alpha-carbon atom of 3,4-dihydroxyacetophenone. Some of the hydrolysis products have previously been obtained from sclerotized pupal cuticle of Manduca sexta [Xu, R., Huang, X., Hopkins, T.L., Kramer, K.J., 1997. Catecholamine and histidyl protein cross-linked structures in sclerotized insect cuticle. Insect Biochemistry and Molecular Biology 27, 101-108; Kerwin, J.L., Turecek, F., Xu, R., Kramer, K.J., Hopkins, T.L., Gatlin, C.L., Yates, J.R., 1999. Mass spectrometric analysis of catechol-histidine adducts from insect cuticle. Analytical Biochemistry 268, 229-237; Kramer, K.J., Kanost, M.R., Hopkins, T.L., Jiang, H., Zhu, Y.C., Xu, R., Kerwin, J.L., Turecek, F., 2001. Oxidative conjugation of catechols with proteins in insect skeletal systems. Tetrahedron 57, 385-392], but the lysine-dihydroxyacetophenone compound and the histidine-DOPALD adduct have not been reported before. It is suggested that the compounds are derived from NADA and NBAD residues which were incorporated into the cuticle during sclerotization, and that the lysine-dihydroxyacetophenone as well as the DOPET and DOPALD containing adducts are degradation products derived from cross-links between the cuticular proteins, whereas the dopamine-containing adducts are derived from a non-crosslinking reaction product.     

Catecholamines and related o-diphenols in the hemolymph and cuticle of the cockroach Leucophaea maderae (F.) during sclerotization and pigmentation

Developmental profiles of catecholamines and related o-diphenols in the hemolymph and cuticle of Leucophaea maderae were determined during sclerotization and pigmentation of last instar nymphs and adults. N-Acetyldopamine (NADA) and dopamine (DA) were the major o-diphenols in hemolymph, whereas 3,4-dihydroxyphenylketoethanol (DOPKET), N-β-alanyldopamine (NBAD), norepinephrine, 3,4-dihydroxyphenylethanol, 3,4-dihydroxyphenylacetic acid, and 3,4-dihydroxyphenylalanine were detected at lower concentrations. The o-diphenols occurred primarily as acid-labile conjugates in hemolymph. Dopamine, conjugated as the 3-O-sulfate ester, and a NADA conjugate(s) were equal in concentration (0.06 mM) in nymphs shortly before adult apolysis. However, NADA increased after adult ecdysis to a peak at 6 h (0.18 mM), while its precursor DA decreased, suggesting N-acetylation of the latter or its metabolism to melanin pigments in the cuticle. In cuticle, NADA, N-acetylnorepinephrine (NANE), DOPKET, and N-β-alanylnorepinephrine (NBANE) accumulated during the early period of adult cuticle sclerotization. DOPKET and NADA (0.4 μmol g⁻¹ each), and NANE (0.2 μmole g⁻¹) occurred at the highest concentrations in tanned adult cuticle. Large amounts of DOPKET conjugates extracted by cold 1.2 M HCl from tanned cuticle which released DOPKET upon hydrolysis at 100°C for 10 min. DA and NBANE (0.2 μmole g⁻¹ each) predominated in tanned nymphal cuticle. Therefore, sclerotization of nymphal cuticle may require more of the N-β-alanyl catecholamines, whereas the adult cuticle contains larger quantities of the N-acetyl derivatives and ketocatechol (DOPKET) metabolites. Black pigmentation of nymphal and adult cuticle occurs during the first few hours after ecdysis, which correlates with relatively high levels of dopamine.     

The conformation of oxytocin in dimethylsulfoxide as revealed by carbon-13 spin-lattice relaxation times

Information was obtained on rates of overall molecular reorientation and segmental motion of amino acid sidechains of oxytocin in dimethylsulfoxide by determination of spin-lattice relaxation times (T₁) at 25 MHz for carbon-13 in natural abundance in the hormone. The T₁ values of the α-carbons of amino acid residues located in the 20-membered ring of oxytocin are all about 50 msec. The overall correlation time for the hormone backbone was estimated to be 8.8 × 10^?10 sec. The sidechains of Tyr, Ile and Gln undergo segmental motion with respect to the backbone of the ring. The T₁ value of the α-carbon of the Leu residue is greater than for any α-carbon in the ring, indicating an increased mobility of the backbone of the C-terminal acyclic peptide as compared to the ring. The β- and γ-carbons of the Pro residue undergo an exo-endo interconversion with regard to the plane formed by α-carbon, δ-carbon and N atom of the Pro pyrollidine ring. These data are discussed in light of results from other experimental and theoretical studies, including carbon-13 spin-lattice relaxation times for oxytocin in aqueous solution.     

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.     

Model sclerotization studies. 3. Cuticular enzyme catalyzed oxidation of peptidyl model tyrosine and dopa derivatives

Incubation of N-acetyltyrosine methyl ester with cuticular enzymes, isolated from the wandering stages of Calliphora sp larvae, resulted in the generation of N-acetyldopa methyl ester when the reaction was carried out in the presence of ascorbate which prevented further oxidation of the o-diphenolic product. Enzymatic oxidation of N-acetyldopa methyl ester ultimately generated dehydro N-acetyldopa methyl ester. The identity of enzymatically produced N-acetyldopa methyl ester and dehydro N-acetyldopa methyl ester has been confirmed by comparison of the ultraviolet and infrared spectral and chromatographic properties with those of authentic samples as well as by nuclear magnetic resonance studies. Since N-acetyldopaquinone methyl ester was also converted to dehydro N-acetyldopa methyl ester and tyrosinase was responsible for the oxidation of N-acetyldopa methyl ester, a scheme for the cuticular phenoloxidase catalyzed conversion of N-acetyltyrosine methyl ester to dehydro N-acetyldopa methyl ester involving the intermediary formation of the quinone and the quinone methide is proposed to account for the observed results. The conversion of N-acetyldopa methyl ester to dehydro derivative remarkably resembles the conversion of the sclerotizing precursor, N-acetyldopamine, to dehydro-N-acetyl-dopamine observed in the insect cuticle. Based on these comparative studies, it is proposed that peptidyl dopa derivatives could also serve as the sclerotizing precursors for the sclerotization of the insect cuticle. © 1995 Wiley-Liss, Inc.     

Oxidation of N-β-alanyldopamine by insect cuticles and its role in cuticular sclerotization

The oxidation products formed when various types of insect cuticle were incubated with N-β-alanyldopamine (NBAD) have been studied by means of reversed phase high performance liquid chromatography, and compared to the corresponding products obtained when N-acetyldopamine (NADA) was incubated with the cuticles. The results indicate that NBAD is oxidized to o-quinone and quinone methide derivatives. In contrast, NADA can be oxidized by some cuticles not only to o-quinone and quinone methide derivatives, but it can also be desaturated to α,β-dehydro-N-acetyldopamine, a probable intermediate in β-sclerotization. Some implications for in vivo sclerotization are discussed.     

Mass spectrometric analysis of catechol-histidine adducts from insect cuticle

Adducts of catechols and histidine, which are produced by reactions of 1,2-quinones and p-quinone methides with histidyl residues in proteins incorporated into the insect exoskeleton, were characterized using electrospray ionization mass spectrometry (ESMS), tandem electrospray mass spectrometry (ESMS-MS, collision-induced dissociation), and ion trap mass spectrometry (ITMS). Compounds examined included adducts obtained from acid hydrolysates of Manduca sexta (tobacco hornworm) pupal cuticle exuviae and products obtained from model reactions under defined conditions. The ESMS and ITMS spectra of 6-(N-3')-histidyldopamine [6-(N-3')-His-DA, pi isomer] isolated from M. sexta cuticle were dominated by a [M + H]+ ion at m/z 308, rather than the expected m/z 307. High-resolution fast atom bombardment MS yielded an empirical formula of C14H18N3O5, which was consistent with this compound being 6-(N-1')-histidyl-2-(3, 4-dihydroxyphenyl)ethanol [6-(N-1')-His-DOPET] instead of a DA adduct. Similar results were obtained when histidyl-catechol compounds linked at C-7 of the catechol were examined; the (N-1') isomer was confirmed as a DA adduct, and the (N-3') isomer identified as an (N-1')-DOPET derivative. Direct MS analysis of unfractionated cuticle hydrolysate revealed intense parent and product ions characteristic of 6- and 7-linked adducts of histidine and DOPET. Mass spectrometric analysis of model adducts synthesized by electrochemical oxidative coupling of N-acetyldopamine (NADA) quinone and N-acetylhistidine (NAcH) identified the point of attachment in the two isomers. A prominent product ion corresponding to loss of CO2 from [M + H]+ of 2-NAcH-NADA confirmed this as being the (N-3') isomer. Loss of (H2O + CO) from 6-NAcH-NADA suggested that this adduct was the (N-1') isomer. The results support the hypothesis that insect cuticle sclerotization involves the formation of C-N cross-links between histidine residues in cuticular proteins, and both ring and side-chain carbons of three catechols: NADA, N-beta-alanyldopamine, and DOPET.     

Reactivity of asparagine residue at the active site of the D105N mutant of fluoroacetate dehalogenase from Moraxella sp. B

Fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX) catalyzes cleavage of the carbon–fluorine bond of fluoroacetate, whose dissociation energy is among the highest found in natural products. Asp105 functions as the catalytic nucleophile that attacks the α-carbon atom of the substrate to displace the fluorine atom. In spite of the essential role of Asp105, we found that site-directed mutagenesis to replace Asp105 by Asn does not result in total inactivation of the enzyme. The activity of the mutant enzyme increased in a time- and temperature-dependent manner. We analyzed the enzyme by ion-spray mass spectrometry and found that the reactivation was caused by the hydrolytic deamidation of Asn105 to generate the wild-type enzyme. Unlike Asn10 of the l-2-haloacid dehalogenase (L-DEX YL) D10N mutant, Asn105 of the fluoroacetate dehalogenase D105N mutant did not function as a nucleophile to catalyze the dehalogenation.     

On the mechanism of formation of N-acetyldopamine quinone methide in insect cuticle

The mechanism of formation of quinone methide from the sclerotizing precursor N-acetyldopamine (NADA) was studied using three different cuticular enzyme systems viz. Sarcophaga bullata larval cuticle, Manduca sexta pharate pupae, and Periplaneta americana presclerotized adult cuticle. All three cuticular samples readily oxidized NADA. During the enzyme-catalyzed oxidation, the majority of NADA oxidized became bound covalently to the cuticle through the side chain with the retention of o-diphenolic function, while a minor amount was recovered as N-acetylnorepinephrine (NANE). Cuticle treated with NADA readily released 2-hydroxy-3′,4′-dihydroxyacetophenone on mild acid hydrolysis confirming the operation of quinone methide sclerotization. Attempts to demonstrate the direct formation of NADA-quinone methide by trapping experiments with N-acetylcysteine surprisingly yielded NADA-quinone-N-acetylcysteine adduct rather than the expected NADA-quinone methide-N-acetylcysteine adduct. These results are indicative of NADA oxidation to NADA-quinone and its subsequent isomerization to NADA-quinone methide. Accordingly, all three cuticular samples exhibited the presence of an isomerase, which catalyzed the conversion of NADA-quinone to NADA-quinone methide as evidenced by the formation of NANE—the water adduct of quinone methide. Thus, in association with phenoloxidase, newly discovered quinone methide isomerase seems to generate quinone methides and provide them for quinone methide sclerotization.     

Detection of protein adduction derived from styrene oxide to cysteine residues by alkaline permethylation

Styrene oxide-cysteine adduction is predominantly involved in protein covalent modification after exposure in vivo to styrene or styrene oxide. In the present study, we developed an alkaline permethylation- and GC/MS-based approach to detect styrene oxide-derived protein adduction. Permethylation of the protein adducts produced two methylthiophenylethanols, namely 2-methylthio-2-phenyl-1-ethanol and 2-methylthio-1-phenyl-1-ethanol. To improve the permethylation efficiency, reaction conditions, including temperature, time, NaOH strength, and molar ratio of CH₃I/NaOH, were explored. Under optimized conditions, the yields of the analyte formation resulting from permethylation of authentic standard α- and β-mercapturic acids, representing α and β isomers of cysteine adducts, were 35% and 28%, respectively. Permethylation of styrene oxide-modified bovine serum albumin released the two methylthiophenylethanols with an α-/β-adduction ratio of 1.5. A concentration-dependent increase in both α- and β-adduction was observed in mouse liver microsomes incubated with styrene at various concentrations. CD-1 mice were administered intraperitoneally with styrene at doses of 0, 50, and 400 mg/kg daily for 5 days. The formation of protein adducts derived from styrene oxide in whole blood in 400 mg/kg group was observed with an α/β ratio of 4.8, suggesting that the reaction of styrene oxide with cysteine residues took place more likely at the α-carbon than the β-carbon of styrene oxide.