Characterization of a new enzyme system that desaturates the side chain of N-acetyldopamine

A novel enzyme system that desaturates the side chain of the catecholamine derivative, N-acetyldopamine (NADA), was isolated and characterized from the larval cuticle of Sarcophaga bullata. The NADA desaturase system which converts NADA to 1,2-dehydro-NADA, surprisingly, does not resemble dehydrogenases such as succinate dehydrogenase. It uniquely performs the desaturation reaction by oxidizing NADA to its corresponding quinone and subsequently converting the resultant quinone to 1,2-dehydro-NADA via NADA quinone methide. Accordingly, desaturase enzyme preparation contained both o-diphenoloxidase activity and NADA quinone:NADA quinone methide isomerase activity. In addition, inhibition studies as well as trapping experiments also confirmed the obligatory formation of NADA quinone as the transient intermediate of the NADA desaturation. It is the first report of a cell-free system causing the side chain desaturation of any catecholamine derivative.     

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.     

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.     

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)     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Enzymic activities involved in the oothecal sclerotization of the praying mantid,Tenodera aridifolia sinensis saussure

The enzyme(s) responsible for the sclerotization of mantid ootheca is secreted by the left colleterial gland. From an extract of the glands of Tenodera aridifolia sinensis, two soluble enzyme fractions of different activities were obtained. One fraction acted on N-acetyldopamine (NADA), a precursor of a representative sclerotizing agent, and produced NADA-quinone. The other did not act on NADA itself but converted the quinone to a highly reactive intermediate, such as quinone methide, which was able to react nonenzymically with nucleophilic compounds. Other insoluble enzyme preparations obtained from the silk and pupal cuticle of the Japanese giant silk moth, Dictyoploca japonica, also had these two activities.     

On the oxidation of 3,4-dihydroxyphenethyl alcohol and 3,4-dihydroxyphenyl glycol by cuticular enzyme(s) from Sarcophaga bullata

The catabolic fate of 3,4-dihydroxyphenethyl alcohol (DHPA) and 3,4-dihydroxyphenylethyl glycol (DHPG) in insect cuticle was determined for the first time using cuticular enzyme(s) from Sarcophaga bullata and compared with mushroom tyrosinase-medicated oxidation. Mushroom tyrosinase converted both DHPA and DHPG to their corresponding quinone derivatives, while cuticular enzyme(s) partly converted DHPA to DHPG. Cuticular enzyme(s)-mediated oxidation of DHPA also accompanied the covalent binding of DHPA to the cuticle. Cuticle-DHPA adducts, upon pronase digestion, released peptides that had bound catechols. 3,4-Dihydroxyphenyl-acetaldehyde, the expected product of side chain desaturation of DHPA, was not formed at all. The presence of N-acetylcysteine, a quinone trap, in the reaction mixture containing DHPA and cuticle resulted in the generation of DHPA-quinone-N-acetylcysteine adduct and total inhibition of DHPG formation. The insect enzyme(s) converted DHPG to its quinone at high substrate concentration and to 2-hydroxy-3′,4′-dihydroxyacetophenone at low concentration. They converted exogenously added DHPA-quinone to DHPG, but acted sluggishly on DHPG-quinone. These results are consistent with the enzymatic transformations of phenoloxidase-generated quinones to quinone methides and subsequent nonenzymatic transformation of the latter to the observed products. Thus, quinone methide formation in insect cuticle seems to be caused by the combined action of two enzymes, phenoloxidase and quinone tautomerase, rather than the action of quinone methide-generating phenoloxidase (Sugumaran: Arch Insect Biochem Physiol 8, 73–88, 1988). It is proposed that DHPA and DHPG in combination can be used effectively to examine the participation of (1) quinone, (2) quinone methide, and (3) dehydro derivative intermediates in the metabolism of 4-alkylcatechols for cuticular sclerotization.     

o-quinone/quinone methide isomerase: a novel enzyme preventing the destruction of self-matter by phenoloxidase-generated quinones during immune response in insects

Melanization and encapsulation of invading foreign organisms observed during the immune response in insects is known to be due to the action of activated phenoloxidase. Phenoloxidase-generated quinones are deposited either directly or after self-polymerization on foreign objects accounting for the observed reactions. Since the reactions of quinones are nonenzymatic, they do not discriminate self from nonself and hence will also destroy self-matter. In this report we present evidence for the presence of a novel quinone/quinone methide isomerase in the hemolymph of Sarcophaga bullata which destroys long-lived quinones and hence acts to protect the self-matter. Quinone methides, formed by the action of this enzyme on physiologically important quinones, being unstable undergo rapid hydration to form nontoxic metabolites.     

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.     

The crystal structure of the ivy Delta4-16:0-ACP desaturase reveals structural details of the oxidized active site and potential determinants of regioselectivity

The multifunctional acyl-acyl carrier protein (ACP) desaturase from Hedera helix (English ivy) catalyzes the Delta(4) desaturation of 16:0-ACP and the Delta(9) desaturation of 18:0-ACP and further desaturates Delta(9)-16:1 or Delta(9)-18:1 to the corresponding Delta(4,9) dienes. The crystal structure of the enzyme has been solved to 1.95 A resolution, and both the iron-iron distance of approximately 3.2A and the presence of a mu-oxo bridge reveal this to be the only reported structure of a desaturase in the oxidized FeIII-FeIII form. Significant differences are seen between the oxidized active site and the reduced active site of the Ricinus communis (castor) desaturase; His(227) coordination to Fe2 is lost, and the side chain of Glu(224), which bridges the two iron ions in the reduced structure, does not interact with either iron. Although carboxylate shifts have been observed on oxidation of other diiron proteins, this is the first example of the residue moving beyond the coordination range of both iron ions. Comparison of the ivy and castor structures reveal surface amino acids close to the annulus of the substrate-binding cavity and others lining the lower portion of the cavity that are potential determinants of their distinct substrate specificities. We propose a hypothesis that differences in side chain packing explains the apparent paradox that several residues lining the lower portion of the cavity in the ivy desaturase are bulkier than their equivalents in the castor enzyme despite the necessity for the ivy enzyme to accommodate three more carbons beyond the diiron site.     

Nonenzymatic transformations of enzymatically generated N-acetyldopamine quinone and isomeric dihydrocaffeiyl methyl amide quinone

We have recently demonstrated that the side chain hydroxylation of N-acetyldopamine and related compounds observed in several insects is caused by a two-enzyme system catalyzing the initial oxidation of catecholamine derivatives and subsequent isomerization of the resultant quinones to isomeric quinone methides, which undergo rapid nonenzymatic hydration to yield the observed products [Saul, S.J. and Sugumaran, M. (1989) FEBS Lett. 249, 155-158]. During our studies on o-quinone/p-quinone methide tautomerase, we observed that quinone methides are also produced nonenzymatically slowly, under physiological conditions. The quinone methide derived from N-acetyldopamine was hydrated to yield N-acetylnorepinephrine as the stable product as originally shown by Senoh and Witkop [(1959) J. Am. Chem. Soc. 81, 6222-6231], while the isomeric quinone methide from dihydrocaffeiyl methylamide exhibited a new reaction to form caffeiyl amide as the stable product. The identity of this product was established by UV and IR spectral studies and by chemical synthesis. We could not find any evidence of intramolecular cyclization of N-acetyldopamine quinone to iminochrome-type compound(s). The importance of quinone methides in these reactions is discussed.     

Contribution of two ζ-carotene desaturases to the poly-cis desaturation pathway in the cyanobacterium Nostoc PCC 7120

The presence of two completely unrelated ζ-carotene desaturases CrtQa and CrtQb in some Nostoc strains is unique. CrtQb is the ζ-carotene desaturase, which was acquired by almost all cyanobacteria. The additional CrtQa can be regarded as an evolutionary relict of the CrtI desaturase present in non-photosynthetic bacteria. By reconstruction of the carotene desaturation pathway, we showed that both enzymes from Nostoc PCC 7120 were active. However, they differed in their preferred utilization of ζ-carotene Z isomers. CrtQa converted ζ-carotene isomers that were poorly metabolized by CrtQb. In this respect, CrtQa complemented the reactions of CrtQb, which is an advantage avoiding dead ends in the poly-cis desaturation pathway. In addition to ζ-carotene desaturation, CrtQa still possesses the Z to E isomerase function of the ancestral desaturase CrtI. Biochemical characterization showed that CrtQb is an enzyme with one molecule of tightly bound FAD and acts as a dehydrogenase transferring hydrogen to oxidized plastoquinone.     

Mechanism of rat liver microsomal stearyl-CoA desaturase. Studies of the substrate specificity, enzyme-substrate interactions, and the function of lipid.

The three purified proteins which are required for microsomal stearyl-CoA desaturation, NADH-cytochrome b5 reductase, cytochrome b5, and desaturase, have been combined with egg lecithin or dimyristyl lecithin vesicles to reconstruct a functional electron transport system capable of utilizing NADH and O2 in the desaturation of stearyl-CoA. Such preparations appear to consist of phospholipid vesicles which contain the three proteins bound to the outer surface of the vesicles. Acyl-CoA derivatives containing 12 to 19 carbon fatty acyl chains are required for desaturase activity while derivatives containing 9 to 20 carbons are capable of binding to the enzyme. Shorter chain acyl-CoA derivatives, free CoA, and free fatty acids do not appear to bind to the enzyme. Inhibition and analog studies suggest that the methylene chain of stearyl-CoA assumes an eclipsed ("gauche") conformation at carbon atoms 9,10 in the enzyme-substrate complex. Furthermore, isotope rate effects obtained with deuterated stearyl-CoA derivatives indicate that hydrogen removal is the rate-limiting step of desaturation. Stearyl-CoA binds to pure liposomes and desaturase-containing liposomes, and it is this form of stearyl-CoA which appears to be the substrate for desaturase. The Arrhenius plots of desaturase activity obtained using desaturase bound to egg lecithin liposomes, in which the liquid crystalline to crystalline phase transition temperature is -5 degrees, was linear between 15 and 35 degrees, while that obtained using desaturase bound to dimyristyl lecithin liposomes showed a break at 24 degrees coinciding with the liquid crystalline to crystalline phase transition temperature for this lipid. The decrease observed in the deuterium isotope rate effect below the transition temperature indicates that a step in the reaction sequence other than hydrogen abstraction becomes rate-limiting when the lipid is in the crystalline state. In this system translational diffusion does not emerge as the rate-limiting step. The liposomes contained sufficient reductase and cytochrome b5 so that translational diffusion was not rate-limiting.