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.     

On the mechanism of side chain oxidation of N-beta-alanyldopamine by cuticular enzymes from Sarcophaga bullata

The metabolism of N-beta-alanyldopamine (NBAD) by Sarcophaga bullata was investigated. Incubation of NBAD with larval cuticular preparations resulted in the covalent bindings of NBAD to the cuticle and generation of N-beta-alanyl-norepinephrine (NBANE) as the soluble product. When the reaction was carried out in presence of a powerful quinone trap viz., N-acetylcysteine, NBANE formation was totally abolished; but a new compound characterized as NBAD-quinone-N-acetylcysteine adduct was generated. These results indicate that NBAD quinone is an obligatory intermediate for the biosynthesis of NBANE in sarcophagid cuticle. Accordingly, phenylthiourea--a well-known phenoloxidase inhibitor--completely inhibited the NBANE production even at 5 microM level. A soluble enzyme isolated from cuticle converted exogenously supplied NBAD quinone to NBANE. Chemical considerations indicated that the enzyme is an isomerase and is converting NBAD quinone to its quinone methide which was rapidly and nonenzymatically hydrated to form NBANE. Consistent with this hypothesis is the finding that NBAD quinone methide can be trapped as beta-methoxy NBAD by performing the enzymatic reaction in 10% methanol. Moreover, when the reaction was carried out in presence of kynurenine, two diastereoisomeric structures of papiliochrome II-(Nar-[alpha-3-aminopropionyl amino methyl-3,4-dihydroxybenzyl]-L-kynurenine) could be isolated as by-products, indicating that the further reactions of NBAD quinone methide with exogenously added nucleophiles are nonenzymatic and nonstereoselective. Based on these results, it is concluded that NBAD is metabolized via NBAD quinone and NBAD quinone methide by the action of phenoloxidase and quinone isomerase respectively. The resultant NBAD quinone methide, being highly reactive, undergoes nonenzymatic and nonstereoselective Michael-1,6-addition reaction with either water (to form NBANE) or other nucleophiles in cuticle to account for the proposed quinone methide sclerotization.     

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.     

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.     

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.     

Studies on the enzymes involved in puparial cuticle sclerotization in Drosophila melanogaster.

The properties of cuticular enzymes involved in sclerotization of Drosophila melanogaster puparium were examined. The cuticle-bound phenoloxidase from the white puparium exhibited a pH optimum of 6.5 in phosphate buffer and oxidized a variety of catecholic substrates such as 4-methylcatechol, N-beta-alanyldopamine, dopa, dopamine, N-acetyldopamine, catechol, norepinephrine, 3,4-dihydroxyphenylglycol, 3,4-dihydroxybenzoic acid, and 3,4-dihydroxyphenylacetic acid. Phenoloxidase inhibitors such as potassium cyanide and sodium fluoride inhibited the enzyme activity drastically, but phenylthiourea showed marginal inhibition only. This result, coupled with the fact that syringaldazine served as the substrate for the insoluble enzyme, confirmed that cuticular phenoloxidase is of the "laccase" type. In addition, we also examined the mode of synthesis of the sclerotizing precursor, 1,2-dehydro-N-acetyldopamine. Our results indicate that this catecholamine derivative is biosynthesized from N-acetyldopamine through the intermediate formation of N-acetyldopamine quinone and N-acetyldopamine quinone methide as established for Sarcophaga bullata [Saul, S. and Sugumaran, M., F.E.B.S. Letters 251, 69-73 (1989)]. Accordingly, successful solubilization and fractionation of cuticular enzymes involved in the introduction of a double bond in the side chain of N-acetyldopamine indicated that they included o-diphenoloxidase, 4-alkyl-o-quinone:p-quinone methide isomerase, and N-acetyldopamine quinone methide:dehydro N-acetyldopamine isomerase and not any side chain desaturase.     

Quinone methides—and not dehydrodopamine derivatives—as reactive intermediates of β-sclerotization in the puparia of flesh fly Sarcophaga bullata

Cuticular phenoloxidase(s) from Sarcophaga bullata larvae oxidized a variety of o-diphenolic compounds. While catechol, 3,4-dihydroxybenzoic acid, dopa, dopamine, and norepinephrine were converted to their corresponding quinone derivatives, other catechols such as 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxyphenethyl alcohol, 3,4-dihydroxyphenyl glycol, 3,4-dihy-droxymandelic acid, and N-acetyldopamine were oxidized to their side-chain oxygenated products. In addition, the enzyme-catalyzed oxidation of the latter group of compounds accompanied the formation of colorless catecholcuticle adducts consistent with the operation of β-sclerotization. Radioactive trapping experiments failed to support the participation of 1,2-dehydro-N-acetyldopamine as a freely formed intermediate during phenoloxidase-mediated oxidation of N-acetyldopamine. When specifically tritiated substrates were provided, cuticular enzyme selectively removed tritium from [7-³H]N-acetyldopamine and not from either [8-³H] or [ring-³H]N-acetyldopamine during the initial phase of oxidation. The above results are consistent with the generation and subsequent reactions of quinone methides as the initial products of enzyme-catalyzed N-acetyldopamine oxidation and confirm our hypothesis that quinone methides and not 1,2-dehydro-N-acetyldopamine are the reactive intermediate of β-sclerotization of sarcophagid cuticle. Quinone methide sclerotization resolves a number of conflicting observations made by previous workers in this field.     

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.     

Quinone and quinone methide as transient intermediates involved in the side chain hydroxylation of N-acyldopamine derivatives by soluble enzymes from Manduca sexta cuticle.

Proteins solubilized from the pharate cuticle of Manduca sexta were fractionated by ammonium sulfate precipitation and activated by the endogenous enzymes. The activated fraction readily converted exogenously supplied N-acetyldopamine (NADA) to N-acetylnorepinephrine (NANE). Either heat treatment (70 degrees C for 10 min) or addition of phenylthiourea (2.5 microM) caused total inhibition of the side chain hydroxylation. If chemically prepared NADA quinone was supplied instead of NADA to the enzyme solution containing phenylthiourea, it was converted to NANE. Presence of a quinone trap such as N-acetylcysteine in the NADA-cuticular enzyme reaction not only prevented the accumulation of NADA quinone, but also abolished NANE production. In such reaction mixtures, the formation of a new compound characterized as NADA-quinone-N-acetylcysteine adduct could be readily witnessed. These studies indicate that NADA quinone is an intermediate during the side chain hydroxylation of NADA by Manduca cuticular enzyme(s). Since such a conversion calls for the isomerization of NADA quinone to NADA quinone methide and subsequent hydration of NADA quinone methide, attempts were also made to trap the latter compound by performing the enzymatic reaction in methanol. These attempts resulted in the isolation of beta-methoxy NADA (NADA quinone methide methanol adduct) as an additional product. Similarly, when the N-beta-alanyldopamine (NBAD)-Manduca enzyme reaction was carried out in the presence of L-kynurenine, two diastereoisomers of NBAD quinone methide-kynurenine adduct (= papiliochrome IIa and IIb) could be isolated.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new mechanism for the control of phenoloxidase activity: inhibition and complex formation with quinone isomerase

Insect phenoloxidases participate in three physiologically important processes, viz., cuticular hardening (sclerotization), defense reactions (immune reaction), and wound healing. Arrest or even delay of any of these processes compromises the survival of insects. Since the products of phenoloxidase action, viz., quinones, are cytotoxic, uncontrolled phenoloxidase action is deleterious to the insects. Therefore, the activity of this important enzyme has to be finely controlled. A novel inhibition of insect phenoloxidases, which serves as a new regulatory mechanism for control of its activity, is described. The activity of phenoloxidases isolated from both Sarcophaga bullata and Manduca sexta is drastically inhibited by quinone isomerase (isolated from Calliphora), an enzyme that utilizes the phenoloxidase-generated 4-alkylquinones. In turn, phenoloxidase reciprocated the inhibition of isomerase. By forming a complex and controlling each other's activity, these two enzymes seem to regulate the levels of endogenously quinones. In support of this contention, an endogenous complex consisting of phenoloxidase, quinone isomerase, and quinone methide isomerase was characterized from the insect, Calliphora. This sclerotinogenic complex was isolated and purified by borate extraction of the larval cuticle, ammonium sulfate precipitation, and Sepharose 6B column chromatography. The complex exhibited a molecular mass of about 620-680 kDa, as judged by size-exclusion chromatography on Sepharose 6B and HPLC and did not even enter 3% polyacrylamide gel during electrophoresis. The phenoloxidase activity of the complex exhibited a wide substrate specificity. Incubation of the complex with N-acetyldopamine rapidly generated N-acetylnorepinephrine, dehydro-N-acetyldopamine, and its dimers. In addition, transient accumulation of N-acetyldopamine quinone was also observed. These results confirm the presence of phenoloxidase, quinone isomerase, and quinone methide isomerase in the complex. Attempts to dissociate the complex with even trace amounts of SDS ended in the total loss of quinone isomerase activity. The complex does not seems to be made up of stoichiometric amounts of individual enzymes as the ratio of phenoloxidase to quinone isomerase varied from preparation to preparation. It is proposed that the complex formation between sequential enzymes of sclerotinogenic pathway is advantageous for the organism to effectively channel various reactive intermediates during cuticular hardening.     

Characterization of quinone tautomerase activity in the hemolymph of Sarcophaga bullata larvae

The hemolymph of Sarcophaga bullata larvae was activated with either zymosan or proteolytic enzymes such as chymotrypsin or subtilisin and assayed for phenoloxidase activity by two different assays. While oxygen uptake studies readily attested to the wide specificty of activated phenoloxidase, visible spectral studies failed to confirm the accumulation of quinone products in the case of 4-alkyl substituted catechols such as N-acetyldopamine and N-β-alanyldopamine. Sepharose 6B column chromatography of the activated hemolymph resolved phenoloxidase activity into two fractions, designated as A and B. Peak A possessed typical o-diphenoloxidase (o-diphenol, oxygen oxidoreductase EC 1.10.3.1) activity, while peak B oxidized physiologically important catecholamine derivatives such as N-acetyldopamine, N-acetylnorepinephrine, and N-β-alanyldopamine into N-acetylnorepinephrine, N-acetylarterenone, and N-β-alanylnorepinephrine, respectively, and converted 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxymandelic acid, and 3,4-dihydroxyphenylglycol into 3,4-dihydroxymandelic acid, 3,4-dihydroxybenzaldehyde, and 2-hydroxy-3′,4′-dihydroxyacetophenone, respectively. These transformations are consistent with the conversion of phenoloxidase-generated quinones to quinone methides and subsequent non-enzymatic transformations of quinone methides. Accordingly, Peak B contained both o-diphenoloxidase activity and quinone tautomerase activity. Sepharose 6B column chromatography of unactivated hemolymph resulted in the separation of quinone tautomerase from prophenoloxidase. The tautomerase rapidly converted both chemically made and mushroom tyrosinase-generated quinones to quinone methides. Thus the failure to observe the accumulation of quinones with N-acyl derivatives of dopamine and related compounds in the whole hemolymph is due to the rapid conversion of these long lived toxic quinones to short lived quinone methides. The latter, being unstable, undergo rapid non-enzymatic transformations to form side-chain-oxygenated products that are non-toxic. The possible roles of quinone isomerase and its reaction products—quinone methides—as essential components of sclerotization of cuticle and defense reaction of Sarcophaga bullata are discussed.     

N-acetyldopamine quinone methide/1,2-dehydro-N-acetyl dopamine tautomerase. A new enzyme involved in sclerotization of insect cuticle

The enzyme system causing the side chain desaturation of the sclerotizing precursor, N-acetyldopamine (NADA), was solubilized from the larval cuticle of Sarcophaga bullata and resolved into three components. The first enzyme, phenoloxidase, catalyzed conversion of NADA to NADA quinone and provided it for the second enzyme (NADA quinone isomerase), which makes the highly unstable NADA quinone methide. Quinone methide was hydrated rapidly and nonenzymatically to form N-acetylnorepinephrine. In addition, it also served as the substrate for the last enzyme, quinone methide tautomerase, which converted it to 1,2-dehydro-NADA. Reconstitution of NADA side chain desaturase activity was achieved by mixing the last enzyme fraction with NADA quinone isomerase, obtained from the hemolymph of the same organism, and mushroom tyrosinase. Therefore, NADA side chain desaturation observed in insects is caused by the combined action of three enzymes rather than the action of a single specific NADA desaturase, as previously thought.     

On the latency and nature of phenoloxidase present in the left colleterial gland of the cockroach Periplaneta americana

The phenoloxidase system responsible for the sclerotization of cockroach ootheca is found to be present as an inactive form in the left colleterial gland of Periplaneta americana. The supernatant fraction obtained by centrifugation of the milky white secretions contained the inactive phenoloxidase which required both sodium dodecyl sulfate (SDS) and the insoluble sediment for exhibiting enzyme activity. Bovine serum albumin could replace the sediment in the activation process. Proteins separated from the supernatant fraction by molecular sieve chromatography on Sephadex G-25 did not require either albumin or the sediment, but required SDS for exhibiting the phenoloxidase activity. Among the detergents tested, SDS (anionic) and cetylpyridinium chloride (cationic) activated the phenoloxidase, but CHAPS (zwitterionic) or nonionic detergents failed to activate the enzyme. The activation caused by SDS occurred well below the critical micellar concentration of SDS indicating that SDS is causing the activation by binding to the protein and altering its conformation. Chloroform-methanol extracts of vestibulum or right gland could replace SDS confirming the presence of endogenous activator(s) of phenoloxidase system. A variety of exogenously added lipids could activate the latent enzyme, among which linoleate, oleate, laurate, linolenate, phosphatidylethanolamine, and phosphatidylglycerol proved to be the effective activators of the latent phenoloxidase. Partially purified phenoloxidase was found to be extremely labile and lost its activity on a) freezing and thawing, b) dialysis, and c) heating for 10 min at 55 degrees C. It exhibited a pH optimum of 7 and was inhibited drastically by phenylthiourea and diethyldithiocarbamate. It readily oxidized a number of o-diphenols such as 3,4-dihydroxybenzylalcohol, 3,4-dihydroxyphenethyl alcohol, catechol, N-acetyldopamine, N-acetylnorepinephrine, dopa, dopamine, etc., but failed to oxidize both 3,4-dihydroxybenzoic acid and 3,4-dihydroxybenzaldehyde. It neither converted the typical laccase substrate syringaldazine to its quinone methide product, nor oxidized the p-diphenols, hydroquinone and methylhydroquinone. Therefore, the enzyme participating in the quinone tanning of cockroach ootheca appears to be a typical o-diphenol oxidase and not a laccase as previously thought.     

Oxidation chemistry of 1,2-dehydro-N-acetyldopamines: direct evidence for the formation of 1,2-dehydro-N-acetyldopamine quinone

Two-electron oxidation of catecholamines either by phenol oxidase or by chemical oxidants such as sodium periodate produces their corresponding o-quinones as observable products. But, in the case of 1,2-dehydro-N-acetyldopamine, an important insect cuticular sclerotizing precursor, phenol oxidase catalyzed oxidation has been reported to generate a quinone methide analog as a transient, but first observable product. ?Sugumaran, M., Semensi, V., Kalyanaraman, B., Bruce, J. M., and Land, E. J. (1992) J. Biol. Chem. 267, 10355-10361. The corresponding quinone has escaped detection until now. However, in this paper, for the first time, we present direct evidence for the formation of dehydro-N-acetyldopamine quinone and show that it can readily be produced from the tautomeric quinone methide imine amide during the chemical oxidation of dehydro-N-acetyldopamine under acidic conditions. This situation is in sharp contrast to other known alkyl-substituted catechol oxidations, where quinone is the first observable product and quinone methide is the subsequently generated product. Dehydro-N-acetyldopamine quinone thus formed is also highly unstable. Semiempirical molecular orbital calculation also indicates that quinone methide imine amide is more stable than the quinone. Chemical considerations indicate that the quinone methide tautomer, and not the dehydro-N-acetyldopamine quinone, is responsible for crosslinking the structural proteins and chitin polymer in the insect cuticle. Therefore, the quinone methide tautomer, and not the quinone, is the key reactive intermediate aiding the hardening of insect cuticle.     

Chemical- and cuticular phenoloxidase- mediated synthesis of cysteinyl–catechol adducts

N-Acetylcysteine adducts of o-benzoquinones derived from catechol, 4-methylcatechol, and N-acetyldopamine were chemically synthesized and characterized by a combination of UV, IR, and NMR spectral studies. Oxidation of catechol, 4-methylcatechol, and N-acetyldopamine by cuticle-bound phenoloxidase from Sarcophaga bullata in the presence of N-acetylcysteine resulted in the formation of covalent adducts between catecholic compounds and N-acetylcysteine. Structural identities of these adducts were established by comparison of their HPLC retention time and UV spectra with those of synthetic adducts and by cochromatography with authentic samples. Although insect cuticle is known to contain only trace amounts of cysteine, the in vitro synthesis of quinone cysteine adducts mediated by cuticular phenoloxidase strongly indicates the occurrence of similar reactions in vivo as well and is in support of Pryor's quinone tanning hypothesis.     

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.     

Quinone methide sclerotization: A revised mechanism for β-sclerotization of insect cuticle

Molecular mechanisms responsible for the stiffening and tanning of insect cuticle are reviewed. Two mechanisms, viz., quinone tanning and β-sclerotization, both involving catecholamine derivatives as sclerotizing precursors, are known to strengthen the cuticle. Quinone tanning mechanism invokes the generation and reactions of o-benzoquinones as the sclerotizing agents, whereas β-sclerotization dictates the activation of catecholamine side chains prior to their incorporation into cuticle. The reactive intermediate for the latter process was proposed by other workers to be 1,2-dehydro-N-acetyldopamine and its quinone. The role of these two compounds in β-sclerotization is critically evaluated. Based on our observation that incubation of cuticular enzyme from Sarcophaga bullata with 4-alkylcatechols results in the production of soluble side chain oxygenated compounds and the formation of catechol-cuticle adducts, an alternate mechanism for β-sclerotization is proposed. This mechanism calls for the generation of quinone methides, tautomers of 4-alkyl-quinones, as the initial products of oxidation of catecholamine derivatives in cuticle. Quinone methides formed spontaneously react with available nucleophiles in cuticle, resulting in the generation of catechol-cuticle adducts and side chain hydroxylated products. Further oxidation of adducts and coupling to other structural units ensure crosslinking of cuticular components. The proposed quinone methide sclerotization accounts for all the chemical observations made on the β-sclerotized cuticle.     

Tyrosinase catalyzes an unusual oxidative decarboxylation of 3,4-dihydroxymandelate

Tyrosinase usually catalyzes the conversion of monophenols to o-diphenols and oxidation of diphenols to the corresponding quinones. However, when 3,4-dihydroxymandelic acid was provided as the substrate, it catalyzed an unusual oxidative decarboxylation reaction generating 3,4-dihydroxybenzaldehyde as the sole product. The identity of the product was confirmed by high-performance liquid chromatography (HPLC) as well as ultraviolet and infrared spectral studies. None of the following enzymes tested catalyzed the new reaction: galactose oxidase, ceruloplasmin, superoxide dismutase, ascorbate oxidase, dopamine beta-hydroxylase, and peroxidase. Phenol oxidase inhibitors such as phenylthiourea, potassium cyanide, and sodium azide inhibited the reaction drastically, suggesting the participation of the active site copper of the enzyme in the catalysis. Mimosine, a well-known competitive inhibitor of tyrosinase, competitively inhibited the new reaction also. 4-Hydroxymandelic acid and 3-methoxy-4-hydroxymandelic acid neither served as substrates nor inhibited the reaction. Putative intermediates such as 3,4-dihydroxybenzyl alcohol and (3,4-dihydroxybenzoyl)formic acid did not accumulate during the reaction. Oxidation to a quinone methide derivative rather than conventional quinone accounts for this unusual oxidative decarboxylation reaction. Earlier from this laboratory, we reported the conversion of 4-alkylcatechols to quinone methides catalyzed by a cuticular phenol oxidase [Sugumaran, M., & Lipke, H. (1983) FEBS Lett. 155, 65-68]. Present studies demonstrate that mushroom tyrosinase will also catalyze quinone methide production with the same active site copper if a suitable substrate such as 3,4-dihydroxymandelic acid is provided.     

Oxidation of 3,4-dihydroxybenzyl alcohol: a sclerotizing precursor for cockroach ootheca.

The oxidation of 3,4-dihydroxybenzyl alcohol, one of the sclerotizing precursors for the tanning of the ootheca of cockroach Periplaneta americana, is reported for the first time. Mushroom tyrosinase catalyzed oxidation of 3,4-dihydroxybenzyl alcohol generated the corresponding quinone which was found to be unstable and readily transformed to produce 3,4-dihydroxybenzaldehyde as the stable product probably through the intermediary formation of a quinone methide. Phenoloxidase isolated from the left collateral gland of P. americana also catalyzed this new reaction. When the enzymatic oxidation of 3,4-dihydroxybenzyl alcohol was performed in the presence of a test protein such as lysozyme, the reactive species formed, caused the oligomerization of test protein. Similar studies with collateral gland proteins, failed to generate oligomers, but produced insoluble polymeric proteins. The probable fate of 3,4-dihydroxybenzyl alcohol for the tanning of cockroach ootheca is discussed.     

4-alkyl-o-quinone/2-hydroxy-p-quinone methide isomerase from the larval hemolymph of Sarcophaga bullata. I. Purification and characterization of enzyme-catalyzed reaction

An enzyme which catalyzes the conversion of certain 4-alkyl-o-benzoquinones to 2-hydroxy-p-quinone methides has been purified to apparent homogeneity from the hemolymph of Sarcophaga bullata by employing conventional protein purification techniques. The purified enzyme migrated with an approximate molecular weight of 98,000 on gel filtration chromatography. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it migrated as a single band with a molecular weight of 46,000, indicating that it is made up of two identical subunits. It exhibited a pH optimum of 6.0 and readily converted chemically synthesized as well as enzymatically generated quinones derived from N-acetyldopamine, N-beta-alanyldopamine, and 3,4-dihydroxyphenethyl alcohol to highly unstable 2-hydroxy-p-quinone methides. The quinone methides thus formed were rapidly and nonenzymatically hydrated to form side chain hydroxylated o-diphenols as the stable product. In support of this proposition, when the enzyme reaction with N-acetyldopamine quinone was conducted in the presence of 10% methanol, racemic beta-methoxy-N-acetyldopamine was recovered as an additional product. The quinones of N-acetylnorepinephrine, N-beta-alanylnorepinephrine, and 3,4-dihydroxyphenylglycol were also attacked by the isomerase, resulting in the formation of N-acetylarterenone, N-beta-alanylarterenone and 2-hydroxy-3',4'-dihydroxyacetophenone, respectively as the stable products. The isomerase converted the dihydrocaffeiyl methyl amide quinone to its quinone methide analog which rapidly tautomerized to yield caffeiyl methyl amide. The importance of quinone isomerase in insect immunity and sclerotization of insect cuticle is discussed.