International Journal of Molecular Sciences

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

Manickam Sugumaran * and Hanine Barek

Department of Biology, University of Massachusetts Boston, Boston, MA 02125, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-617-287-6598

Academic Editor: Andrzej Slominski Received: 16 September 2016; Accepted: 12 October 2016; Published: 20 October 2016

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

Keywords: melanogenesis; sclerotization; tyrosinase; phenoloxidase; laccase; dopachrome tautomerase; eumelanin; dopamine; dopa

1. Introduction Melanins are a group of widely distributed phenolic biopolymers arising from the amino acid, tyrosine and related phenolic compounds [1–3]. They are responsible for the skin, hair (or coat or feather), and eye color in animals. Plants as well as fungi and even some bacteria synthesize melanin not only from tyrosine but also from a variety of different phenolic compounds. Animals synthesize three different kinds of melanin viz., pheomelanin, eumelanin and neuromelanin by the action of ubiquitously present enzyme tyrosinase as well as phenoloxidases. The yellow to red pigment found in the red hair, feather and fur of animals is due to pheomelanin. Pheomelanin is produced by the oxidative polymerization of cysteinyldopa, which in turn is synthesized by the coupling of enzymatically generated dopaquinone with the amino acid, cysteine. The brown to black eumelanins are formed by the oxidative polymerization of dihydroxyindoles that are derived from dopa and its derivatives. Neuromelanin is produced in substantia nigra and is made up of dopamine derived polymeric materials. Studies on the biosynthesis, properties, function and structure of melanins have been thoroughly examined by numerous research groups worldwide for several decades [1–9]. However, several aspects of melanin biochemistry including biosynthesis, metabolism, structure, biological and molecular biological aspects of the enzymes associated with the process are yet to be unraveled. The focus of this article will be limited to the biosynthesis, some functions and comparative biochemistry of melanogenesis and related processes in insects and higher animals.

Int. J. Mol. Sci. 2016, 17, 1753; doi:10.3390/ijms17101753 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2016, 17, 1753 2 of 24

2. Raper–Mason Pathway for the Biosynthesis of Eumelanin Int. J. Mol. Sci. 2016, 17, 1753 2 of 24 Classical studies carried out on melanin biosynthesis about a century ago by Raper and his associates2. Raper–Mason established Pathway the intermediacy for the Biosynthesis of dopa, 5,6-dihydroxyindoleof Eumelanin (DHI) and 5,6-dihydroxyindole- 2-carboxylicClassical acid (DHICA)studies carried in eumelanin out on melanin biosynthesis biosynthesis [10 about–12]. a Usingcentury partiallyago by Raper purified and his enzyme preparationsassociates from established the mealworm the intermediacyTenebrio of molitordopa, 5,6-dihydroxyindole, Raper oxidized (DHI) tyrosine and to5,6-dihydroxy the red colored (dopachrome)indole-2-carboxylic stage and acid allowed (DHICA) the reactionin eumelanin to undergo biosynthesis further [10–12]. transformation. Using partially From purified this reaction mixture,enzyme he isolated preparations and characterized from the mealworm DHI as Tenebrio the major molitor product,, Raper and oxidized DHICA tyrosine as the to minor the red product. Therefore,colored he (dopachrome) deduced that stage the oxidativeand allowed polymerization the reaction to undergo of DHI alongfurther with transformation. minor amount From of this DHICA reaction mixture, he isolated and characterized DHI as the major product, and DHICA as the minor polymerization leads to the production of insoluble melanin pigment [10–12]. Later Mason identified product. Therefore, he deduced that the oxidative polymerization of DHI along with minor amount dopachrome as the red pigment and confirmed its conversion to DHI [13,14]. He also demonstrated of DHICA polymerization leads to the production of insoluble melanin pigment [10–12]. Later the two-electronMason identified oxidation dopachrome of catechols as the to red quinones. pigment Thus,and confirmed the successful its conversion identification to DHI of[13,14]. dopaquinone, He dopachrome,also demonstrated DHI and DHICAthe two-electron as an important oxidation intermediates of catechols ofto eumelanogenesis,quinones. Thus, the established successful by the pioneeringidentification work of of Raper dopaquinone, and Mason dopachrome, led to the DHI proposal and DHICA shown as inan Figureimportant1 for intermediates the biosynthesis of of melanin.eumelanogenesis, This simplistic established view was by accepted the pioneering widely. Evenwork thoughof Raper the and proposal Mason acknowledgesled to the proposal the minor role ofshown DHICA, in Figure it assumes 1 for the that biosynthesis DHI is the of major melanin. precursor This simplistic for melanin view inwas animals. accepted After widely. dopaquinone Even formation,though the the rest proposal of the reactions acknowledges in this the pathway minor role could of proceedDHICA, nonenzymaticallyit assumes that DHI and is the spontaneously major precursor for melanin in animals. After dopaquinone formation, the rest of the reactions in this without the need for any enzyme resulting in dark colored insoluble melanin pigment in test tubes. pathway could proceed nonenzymatically and spontaneously without the need for any enzyme Therefore,resulting for ain long dark time, colored the insoluble common melanin understanding pigment isin that test tyrosinasetubes. Therefore, is the solefor a enzymelong time, responsible the for melanincommon biosynthesis. understanding is that tyrosinase is the sole enzyme responsible for melanin biosynthesis.

Figure 1. Raper–Mason pathway for the biosynthesis of melanin. The bifunctional enzyme, Figure 1. Raper–Mason pathway for the biosynthesis of melanin. The bifunctional enzyme, tyrosinase (A) converts tyrosine and dopa to dopaquinone. Dopaquinone undergoes instantaneous tyrosinase (A) converts tyrosine and dopa to dopaquinone. Dopaquinone undergoes instantaneous intramolecular nonenzymatic cyclization forming leucochrome, which is rapidly oxidized by intramoleculardopaquinone nonenzymatic to dopachrome. cyclization The red colored forming dopachrome leucochrome, is converted which to is5,6-dihydroxyindole rapidly oxidized by dopaquinone(DHI) as tothe dopachrome. major product Theand red5,6-dihydroxy colored dopachromeindole-2-carboxylic is converted acid (DHICA) to 5,6-dihydroxyindole as the minor (DHI)product. as the major Oxidative product polymerization and 5,6-dihydroxyindole-2-carboxylic of dihydroxyindoles produces the acid mela (DHICA)nin pigment. as the Tyrosinase minor product. Oxidativeis assumed polymerization to be the sole of dihydroxyindolesenzyme associated with produces this pathway the melanin and the pigment. rest of the Tyrosinase reactions (B) is assumedare to be thepresumed sole enzyme to be of associated nonenzymatic with origin. this pathway and the rest of the reactions (B) are presumed to be of nonenzymatic origin. 3. Discovery of Dopachrome Tautomerase 3. DiscoveryThe ofpopular Dopachrome belief that Tautomerase tyrosinase is the only enzyme associated with melanogenesis was shattered with the discovery of dopachrome converting enzyme in 1980s [15,16]. Pawelek and Theassociates popular discovered belief that a tyrosinase new enzyme is the in only mammal enzymeian associatedsystem that with bleached melanogenesis the red color was shatteredof with thedopachrome discovery and of called dopachrome this enzyme converting as dopachro enzymeme-converting in 1980s enzyme [15 ,or16 dopachrome]. Pawelek conversion and associates discoveredfactor. a The new product enzyme of inthe mammalian reaction was systeminitially thatidentified bleached as DHI. the Purification red color of and dopachrome characterization and called this enzymeof this asnew dopachrome-converting enzyme proved to be extremely enzyme difficul or dopachromet as it is always conversion found to factor. be associated The product with of the reactiontyrosinase was initially [17]. identifiedSubsequently, asDHI. various Purification group of andworkers characterization characterized of the this enzyme new enzyme devoid proved of to tyrosinase and demonstrated that the reaction catalyzed by this enzyme is dopachrome conversion be extremely difficult as it is always found to be associated with tyrosinase [17]. Subsequently, various group of workers characterized the enzyme devoid of tyrosinase and demonstrated that the reaction catalyzed by this enzyme is dopachrome conversion to DHICA [18–20]. Therefore, this enzyme Int. J. Mol. Sci. 2016, 17, 1753 3 of 24 is named as dopachrome isomerase [18] or dopachrome tautomerase [19,20]. However, the most widely accepted and currently used name for this enzyme is dopachrome tautomerase (DCT) [19,20].

In insectsInt. [J.21 Mol.–23 Sci.] 2016 and, 17 cuttlefish, 1753 [24], a different enzyme that catalyzes the conversion of dopachrome3 of 24 to DHI was characterized. to DHICA [18–20]. Therefore, this enzyme is named as dopachrome isomerase [18] or dopachrome 4. Insecttautomerase Dopachrome [19,20]. Decarboxylating However, the most Enzymewidely accept Is Alsoed and a Tautomerasecurrently used name for this enzyme is dopachrome tautomerase (DCT) [19,20]. In insects [21–23] and cuttlefish [24], a different enzyme that Ascatalyzes stated above, the conversion insect dopachromeof dopachrome conversionto DHI was characterized. enzyme produces DHI as the product [21–23]. Therefore, many scientists wondered if this enzyme should also be called dopachrome tautomerase. However,4. Insect a careful Dopachrome examination Decarboxylating of the Enzyme substrate Is Also specificity a Tautomerase of the enzyme revealed that the insect dopachromeAs stated above, conversion insect dopachrome factor is indeed conversion a tautomerase enzyme produces [23]. DHI Insect as the enzyme product [21–23]. exhibits wide substrateTherefore, specificity many and scientists attacks wondered a number if this of Lenzyme-dopachrome should also derivatives, be called dopachrome but not D -dopachromestautomerase. [23]. However, a careful examination of the substrate specificity of the enzyme revealed that the insect If L-dopachrome is provided as the substrate, it readily decarboxylates the substrate and produces dopachrome conversion factor is indeed a tautomerase [23]. Insect enzyme exhibits wide substrate DHI. Similarly if α-methyl dopachrome is provided, it generates 2-methyl DHI (Figure2). Therefore, specificity and attacks a number of L-dopachrome derivatives, but not D-dopachromes [23]. If the insectL-dopachrome enzyme appears is provided to be as a the decarboxylase. substrate, it re However,adily decarboxylates surprisingly, the substrate it also attacksand produces dopachrome methylDHI. ester Similarly producing if α-methyl its isomeric dopachrome product, is provided, DHICA it methylgenerates ester 2-methyl (Figure DHI2 (Figure). In fact, 2). Therefore, this compound serves asthe a insect better enzyme substrate appears than to dopachromebe a decarboxylase. or α However,-methyldopachrome surprisingly, it foralso the attacks insect dopachrome enzyme. In this instance,methyl it behaves ester producing very much its isomeric like the product, mammalian DHICA enzymemethyl ester in catalyzing(Figure 2). In thefact, simple this compound isomerization reaction.serves What as a is better even substrate more interestingthan dopachrome is its or ability α-methyldopachrome to attack α-methyldopachrome for the insect enzyme. methylIn this ester, instance, it behaves very much like the mammalian enzyme in catalyzing the simple isomerization α which canreaction. neither What undergo is even deprotonationmore interesting atis its the ability-carbon to attack atom α-methyldopachrome nor decarboxylation. methyl However, ester, this compoundwhich is can converted neither undergo into a stabledeprotonation quinone at the methide α-carbon and atom is nor discharged decarboxylation. from the However, active this site as the sole productcompound (Figure is converted2). These into studies a stable not quinone only established methide and the is discharged transient formationfrom the active of quinone site as the methide as a reactivesole product intermediate (Figure during2). These melanin studies biosynthesis,not only established but also the confirmedtransient formation that the of insect quinone enzyme is indeed anmethide isomerase as a reactive [23]. Thus,intermediate the insect during enzyme melanin catalyzes biosynthesis, the decarboxylationbut also confirmed of that some the dopachromeinsect enzyme is indeed an isomerase [23]. Thus, the insect enzyme catalyzes the decarboxylation of some derivatives and tautomerization of others. To distinguish it from the mammalian DCT, this enzyme dopachrome derivatives and tautomerization of others. To distinguish it from the mammalian DCT, can be designatedthis enzyme can asdopachrome be designated as decarboxylase/tautomerase dopachrome decarboxylase/tautomerase (DCDT). (DCDT).

Figure 2. Reactions catalyzed by insect dopachrome conversion factor. Insect dopachrome Figure 2. Reactions catalyzed by insect dopachrome conversion factor. Insect dopachrome conversion conversion factor catalyzes decarboxylative rearrangement of dopachrome to DHI. It also accepts α factor catalyzesα-methyl dopachrome decarboxylative as the rearrangement substrate and produc of dopachromees 2-methyl toDHI DHI. as the It alsosole acceptsproduct. If-methyl dopachromedopachrome as the methyl substrate ester andis provided produces as the 2-methyl substrate, DHI it uniquely as the co solenverts product. it to DHICA If dopachrome methyl ester, methyl ester isthus provided acting as as a thetypical substrate, tautomerase. it uniquely Finally with converts α-methyl it todopachrome DHICA methyl ester, ester, which thus can acting as a typicalneither tautomerase. undergo decarboxylation Finally with α -methylnor deprotonation dopachrome at α-carbon methyl atom, ester, the which enzyme can neithercauses an undergo decarboxylationisomerization nor producing deprotonation the stable at quinoneα-carbon methide. atom, the enzyme causes an isomerization producing the stable quinone methide. 5. Mechanism of Dopachrome Conversion Reaction

5. MechanismEarlier of Dopachromestudies advocated Conversion the intermediacy Reaction of an indolenine derivative for dopachrome conversion reaction (Figure 3) [25]. However the indolenine route assumes electron donating Earlier studies advocated the intermediacy of an indolenine derivative for dopachrome conversion reaction (Figure3)[ 25]. However the indolenine route assumes electron donating properties for the Int. J. Mol. Sci. 2016, 17, 1753 4 of 24

Int. J. Mol. Sci. 2016, 17, 1753 4 of 24 C=N in dopachrome. In reality it is electron withdrawing in the direction of C=N bond. Indeed, it is this propertyproperties that makes for the dopachrome C=N in dopachrome. stable as In the reality two groupsit is electron C=O withdrawing and C=N exhibit in the opposite direction polarityof C=N and stabilizebond. the Indeed, dopachrome it is this structure. property Thisthat routemakes also dopachrome generates stable two differentas the two indolenine groups C=O derivatives—one and C=N that retainsexhibit theopposite carboxyl polarity group and and stabilize the other the dopach that hasrome undergone structure. decarboxylation This route also generates (Figure3). two On the different indolenine derivatives—one that retains the carboxyl group and the other that has other hand, our group first showed that quinone methide is the transient intermediate formed during undergone decarboxylation (Figure 3). On the other hand, our group first showed that quinone melanogenesismethide is boththe transient in chemical intermediate reaction formed and in during enzymatic melanogenesis reactions both [23,26 in] chemical by demonstrating reaction and the in facile isomerizationenzymatic ofreactionsα-methyl [23,26] dopachrome by demonstrating methyl ester the facile to a stable isomerization quinone of methide α-methyl that dopachrome can be isolated and characterizedmethyl ester to [a26 stable]. Subsequently, quinone methide Prota that and can his be associates isolated and also characterized confirmed [26]. the Subsequently, quinone methide formationProta bothand his by associates characterizing also confirmed the quinone the quinone methide methide produced formation from α both-methyl by characterizing dopachrome the methyl esterquinone and by demonstrating methide produced primary from α kinetic-methyl isotope dopachrome effect withmethyl the ester 3,3-dideuterated and by demonstrating dopachrome primary [27 ,28]. As shownkinetic in isotope Figure 3effect, the decarboxylationwith the 3,3-dideuterated will be thedopachrome rate-determining [27,28]. As step shown for DHI in Figure production 3, the from indoleninedecarboxylation route. Hence will be substitutionthe rate-determining of deuterium step for atDHI 3-position production should from indolenine have no route. effect Hence on rate of conversionsubstitution of dopachrome of deuterium to at DHI. 3-position However, should a largehave no kinetic effect isotopeon rate of effect conversion is observed of dopachrome for this reaction to DHI. However, a large kinetic isotope effect is observed for this reaction thereby discounting the thereby discounting the indolenine route [27]. For DHICA production also, indolenine route will be indolenine route [27]. For DHICA production also, indolenine route will be less favorable than the less favorablequinone methide than the route quinone because methide the rate route determinin becauseg thestep rate would determining be removal step of wouldproton befrom removal the of proton2-position from the and 2-position not from and3-position. not from 3-position.

Figure 3. Two possible routes for the conversion of dopachrome. Dopachrome can undergo either (a) Figure 3. Two possible routes for the conversion of dopachrome. Dopachrome can undergo deprotonation or (b) decarboxylation to generate two different indolenine derivatives. Rapid either (a) deprotonation or (b) decarboxylation to generate two different indolenine derivatives. aromatization of these unstable intermediates will generate DHICA and DHI respectively. However, Rapid aromatization of these unstable intermediates will generate DHICA and DHI respectively. this route is unlikely to operate. On the other hand, a common quinone methide intermediate formed However,by isomerization this route is of unlikely dopachrome to operate. can either On the lose other proton hand, (route a common a′) or quinonecarboxyl methidegroup (route intermediate b′) 0 0 formeddepending by isomerization upon the reaction of dopachrome conditions canproducing either loseDHI protonor DHICA. (route a ) or carboxyl group (route b ) depending upon the reaction conditions producing DHI or DHICA. A single quinone methide intermediate can account for both DHI production and DHICA Aproduction single quinone (Figure 3). methide This route intermediate will also ackn canowledge account the for large both primary DHI productionkinetic isotope and effect DHICA observed between the protium form and the deuterium form of the 3-substituted dopachrome [27]. production (Figure3). This route will also acknowledge the large primary kinetic isotope effect Quantum chemical calculations of copper chelated dopachrome also supports this contention and observed between the protium form and the deuterium form of the 3-substituted dopachrome [27]. confirms that deprotonation of the β-proton is energetically most favorable leading to quinone Quantummethide chemical production calculations [29]. Once of formed, copper chelatedquinone methide dopachrome can undergo also supports two different this contention reactions. and confirmsDecarboxylation that deprotonation coupled of aromatization the β-proton of is the energetically quinonoid mostring will favorable produce leading DHI as to quinonethe product. methide productionDecarboxylation [29]. Once of formed,quinone quinonemethide is methide naturally can a facile undergo reaction two as different it is the β reactions.,γ-unsaturated Decarboxylation carboxy coupledcompound. aromatization Nondecarboxylative of the quinonoid isomerization ring willwill producegenerate DHICA DHI as as the the product.product. Both Decarboxylation reactions of quinoneseem to methideoccur in nature. is naturally Even with a facile the enzyme reaction-catalyzed as it is thereaction,β,γ-unsaturated mammalian DCT carboxy takes compound.up the Nondecarboxylativenondecarboxylative isomerization tautomerization will route generate producin DHICAg DHICA as the as product. the product Both [18–20], reactions and seemthe insect to occur in nature.DCDT Even produces with theDHI enzyme-catalyzed as the product [21–23]. reaction, With mammalian nonenzymatic DCT takesreaction, up thedepending nondecarboxylative on the tautomerization route producing DHICA as the product [18–20], and the insect DCDT produces DHI as the product [21–23]. With nonenzymatic reaction, depending on the presence or absence of bivalent Int. J. Mol. Sci. 2016, 17, 1753 5 of 24 metal ions the ratio of DHICA to DHI seems to vary [25]. In chemical conversions of dopachrome carried out at pH 6.5, DHI is produced as the major product. When the pH was changed to 13, DHICA is producedInt. J. Mol. as Sci. the 2016 major, 17, 1753 product under the same conditions thereby showing the influence5 of of 24 pH on the rearrangement of dopachrome [30]. presence or absence of bivalent metal ions the ratio of DHICA to DHI seems to vary [25]. In chemical 6. Dopachromeconversions Conversion—5,6-Dihydroxyindoleof dopachrome carried out at pH 6.5, DHI (DHI) is produced versus 5,6-Dihydroxyindole-2-carboxylicas the major product. When the Acid (DHICA)pH was changed to 13, DHICA is produced as the major product under the same conditions thereby showing the influence of pH on the rearrangement of dopachrome [30]. To best of our knowledge, all known insect dopachrome DCDTs convert dopachrome to DHI and all mammalian6. Dopachrome DCTs Conversion—5,6-D produce DHICAihydroxyindole as the product. (DHI) Why versus is there such a marked difference exhibited by these5,6-Dihydroxy two enzymes indole-2-carboxylic which act on the Acid same (DHICA) substrate? Different explanations are possible to account for this difference.To best of Theour firstknowledge, one is relatedall known to insect the metal dopachrome ion present DCDTs at theconvert active dopachrome site. The to mammalian DHI DCT isand a metalloprotein all mammalian DCTs having produce zinc at DHICA its active as the site prod [31,uct.32]. Why Therefore, is there itsuch is possible a marked that difference this enzyme mimicsexhibited the nonenzymatic by these two enzymes bivalent which metal act ion on assisted the same dopachromesubstrate? Different conversion explanations producing are possible DHICA as the productto account [25 ].for The this naturedifference. of theThe metalfirst one ion is rela presentted to atthe the metal active ion present site of insectat the active DCDT site. is The yet to be determined.mammalian If metal DCT is ions a metalloprotein were absent having at the zinc active at its site,active insect site [31,32]. enzyme Therefore, reaction it is would possible mirror that the this enzyme mimics the nonenzymatic bivalent metal ion assisted dopachrome conversion nonenzymatic conversion of dopachrome to DHI [25]. Insect enzyme might eject quinone methide producing DHICA as the product [25]. The nature of the metal ion present at the active site of insect α intermediateDCDT is from yet to the be activedetermined. site as If observedmetal ions inwere the absent case ofat the-methyl active site, dopachrome insect enzyme methyl reaction ester [26]. The ejectedwould quinone mirror the methide nonenzymatic being aconversionβ,γ-unsaturated of dopachrome acid, will to DHI undergo [25]. Insect spontaneous enzyme decarboxylation,might eject generatingquinone DHI methide (Figure intermediate3). If the from metal the ionactive present site as observed in insect in enzyme the case of is α also-methyl zinc, dopachrome then alternate explanationsmethyl haveester to[26]. be The considered. ejected quinone methide being a β,γ-unsaturated acid, will undergo Anspontaneous interesting decarboxylation, aspect about genera the natureting DHI of metal(Figure binding 3). If the metal site present ion present on mammalianin insect enzyme DCT is is its remarkablealso zinc, similarity then alternate to the explanations copper-binding have to site be of considered. tyrosinase [32]. While binuclear copper is essential An interesting aspect about the nature of metal binding site present on mammalian DCT is its for oxygen activation, no such binuclear center is needed for dopachrome isomerization. Therefore, it remarkable similarity to the copper-binding site of tyrosinase [32]. While binuclear copper is is reasonable to assume that one zinc atom is bound to the quinonoid side and the other to the imine essential for oxygen activation, no such binuclear center is needed for dopachrome isomerization. groupTherefore, and the carboxylit is reasonable group to asassume shown that in one Figure zinc atom4. This is bound might to stabilize the quinonoid the intermediate side and the other quinone methideto andthe imine willallow group its and rearrangement the carboxyl togroup DHICA. as shown The insectin Figure enzymes 4. This do might not show stabilize any sequencethe homologyintermediate to mammalian quinone methide counterpart and will or evenallow toits insectrearrangement phenoloxidases. to DHICA. Bioinformatics The insect enzymes analysis do did not revealnot show any kindany ofsequence homology homology even atto themammalian metal binding counterpart site between or even theto twoinsect classes phenoloxidases. of dopachrome convertingBioinformatics enzymes. analysis Insect did DCDTs not reveal may any not kind have of thehomology same kindeven at of the two metal metal binding binding site sitesbetween on them. They maythe two have classes one of metal dopachrome ion (or converting none) and enzymes. might allowInsect DCDTs the quinone may not methide have the to same leave kind the of active two metal binding sites on them. They may have one metal ion (or none) and might allow the site. The released quinone methide then undergoes nonenzymatic decarboxylative transformation quinone methide to leave the active site. The released quinone methide then undergoes producingnonenzymatic DHI in thisdecarboxylative case. transformation producing DHI in this case.

Figure 4. One possible mechanism for dopachrome conversion to DHICA by mammalian Figure 4. One possible mechanism for dopachrome conversion to DHICA by mammalian dopachrome dopachrome tautomerase (DCT). Mammalian DCT possesses two metal binding sites. If one metal tautomerase (DCT). Mammalian DCT possesses two metal binding sites. If one metal ion binding ion binding site is occupied by the quinonoid nucleus and the other by the amino group and carboxyl site isgroup, occupied it would by theprevent quinonoid the spontaneous nucleus decarbo and thexylation other of by quinone the amino methide group intermediate and carboxyl thereby group, it wouldcausing prevent the isomerization the spontaneous to DHICA decarboxylation. of quinone methide intermediate thereby causing the isomerization to DHICA.

Int. J. Mol. Sci. 2016, 17, 1753 6 of 24

Finally, the amino acid residues present at the active site could also cause the differences in the course ofInt. the J. Mol. reaction. Sci. 2016, 17 For, 1753 instance, if the carboxyl group of dopachrome is situated close to6 of the 24 amino group of lysine or imidazole group of histidine, or guanido group of arginine present at the active site Finally, the amino acid residues present at the active site could also cause the differences in the of the enzyme, it will be protected from decarboxylation by strong ionic interactions. The guanido course of the reaction. For instance, if the carboxyl group of dopachrome is situated close to the group ofamino arginine group hasof lysine the idealor imidazol geometrye group that of histidine, is suitable or guanido for binding group theof arginine carboxyl present group. at the This will stabilizeactive the quinonesite of the methideenzyme, it intermediatewill be protected and from favor decarboxylation the isomerization by strong reaction ionic interactions. leading The to DHICA productionguanido in the group case of of arginine mammalian has the DCTideal asgeometry shown that in Figureis suitable5. The for bindingslaty mutant the carboxyl that isgroup. defective in the activityThis will of dopachrome stabilize the quinone isomerase methide has intermediate just one amino and favor acid the modification. isomerization Argininereaction leading in this to protein is modifiedDHICA to production glutamine in (Arg the case194 →of mammGln),alian which DCT is as solely shown responsible in Figure 5. The for theslaty drasticmutant that reduction is in defective in the activity of dopachrome isomerase has just one amino acid modification. Arginine in the activity of the enzyme [33,34]. This strongly suggests the critical role played by the arginine this protein is modified to glutamine (Arg194 → Gln), which is solely responsible for the drastic residuereduction in catalysis in the and/or activity binding of the enzyme of substrate. [33,34]. This Lack strongly of such suggests a group the or critical presence role played of a carboxyl by the group could causearginine the residue ejection in ofcatalysis quinone and/or methide binding from of su thebstrate. active Lack site of after such dopachrome a group or presence isomerization of a and subsequentcarboxyl nonenzymatic group could cause decarboxylation the ejection of in quinon the casee methide of insect from DCDT the active (Figure site after5). Unfortunately, dopachrome not much informationisomerization is and available subsequent on the nonenzymatic structure of decarboxylation the insect enzyme in the tocase assess of insect the DCDT role of (Figure amino 5). acid side Unfortunately, not much information is available on the structure of the insect enzyme to assess the chain in altering the course of the reaction. Nevertheless, it is imperative that the active site geometry role of amino acid side chain in altering the course of the reaction. Nevertheless, it is imperative that has a decisivethe active role site in geometry diverting has thea decisive reaction role either in diverting to DHICA the reaction or in either favor to of DHICA DHI. Furtheror in favor studies of are desperatelyDHI. neededFurther studies to resolve are desperately this aspect. needed to resolve this aspect.

Figure 5. Possible role of active site in diverting the course of dopachrome conversion reaction. The Figure 5. Possible role of active site in diverting the course of dopachrome conversion reaction. crucial arginine reside at the metal binding site identified in the case of mammalian DCT might bind The crucialto the arginine carboxyl residegroup and atthe protect metal it from binding decarbo sitexylation identified reaction, in thethus case allowing of mammalian the production DCT of might bind tothe sole carboxyl product, group DHICA. and Insect protect dopachrome it from decarboxylationdecarboxylase/tautomerase reaction, (DCDT) thus allowingmay not have the such production of the solea protective product, mechanism DHICA. and Insect might dopachrome even have a carbo decarboxylase/tautomerasexyl group that will favor the decarboxylation (DCDT) may of not have such a protectivequinone methide mechanism to DHI. and might even have a carboxyl group that will favor the decarboxylation of quinone methide to DHI. 7. DHICA Oxidase

7. DHICAMammalian Oxidase melanogenesis seems to be aided by at least three different proteins. They are tyrosinase, tyrosine-related protein 2, which is dopachrome tautomerase, and tyrosinase-related Mammalianprotein 1 or melanogenesisDHICA oxidase. The seems genes to designated be aided as byalbino at, leastslaty, and three brown different, respectively, proteins. code for They are tyrosinase,these tyrosine-relatedthree proteins in mouse protein [33–3 2,7]. whichDHICA turned is dopachrome out to be a poor tautomerase, substrate for and mouse tyrosinase-related tyrosinase proteinand 1 or hence, DHICA a successful oxidase. search The geneslead to designatedthe discovery as ofalbino DHICA, slaty oxidase, and ofbrown brown, respectively,loci as the third code for enzyme in melanogenic pathway [36,37]. In humans, however, tyrosinase seems to exhibit broader these three proteins in mouse [33–37]. DHICA turned out to be a poor substrate for mouse tyrosinase substrate specificity and is able to oxidize DHICA [38]. A 100-kDa membrane protein called Pmel and hence,17/silver a successful was also identified search leadfrom human to the cell discovery lines that of is DHICAable to catalyze oxidase the conversion of brown of loci DHICA as the third enzymeto in melanin melanogenic [39]. DHICA pathway oxidase [36 is, 37able]. Into humans,form heterodimeric however, structure tyrosinase with seemstyrosinase to exhibit[40]. The broader substratemetal specificity ion present and at isthe able active to site oxidize of this DHICA enzyme needs [38]. Ato 100-kDabe identified. membrane More work protein is needed called to Pmel 17/silvershed was light also on identified the structure, from functi humanon and cell mechanism lines that of DHICA is able tooxidase. catalyze the conversion of DHICA to melanin [39]. DHICA oxidase is able to form heterodimeric structure with tyrosinase [40]. The metal ion present at the active site of this enzyme needs to be identified. More work is needed to shed light on the structure, function and mechanism of DHICA oxidase. Int. J. Mol. Sci. 2016, 17, 1753 7 of 24 Int. J. Mol. Sci. 2016, 17, 1753 7 of 24

8. Modified Modified Raper–Mason Raper–Mason Pathway Pathway for for the the Biosynthesis Biosynthesis of of Eumelanin Eumelanin Eventually all these discoveries led to the modificationmodification of the Raper–Mason pathway as shown in FigureFigure6 .6. AA notenote onon thethe tyrosinase tyrosinase reaction reaction should should be be mentioned mentioned here. here. DetailedDetailed mechanistic mechanistic studies revealed that that tyrosinase tyrosinase possess possess two two differen differentt activities. activities. The The first first activity activity is responsible is responsible for forthe theconversion conversion of oftyrosine tyrosine to todopaquinone dopaquinone dire directlyctly [41]. [41]. The The second second activity activity is is the typicaltypical o-diphenoloxidase activity that causescauses thethe oxidationoxidation ofof catecholscatechols toto oo-quinones.-quinones. ThusThus tyrosinasetyrosinase converts tyrosine as wellwell dopa to dopaquinone. Intramolecular cyclization of the dopaquinone leads to thethe generation generation of colorlessof colorless leucochrome, leucochrome, which which is further is oxidizedfurther byoxidized dopaquinone by dopaquinone to dopachrome to thatdopachrome accumulates that in accumulates the reaction in mixture. the reaction During mixtur this process,e. During dopaquinone this process, itself dopaquinone gets reduced itself to dopa, gets whichreduced is to further dopa, oxidized which is byfurther tyrosinase oxidized to dopaquinone.by tyrosinase to Mammalian dopaquinone. DCT Mammalian converts dopachrome DCT converts to DHICAdopachrome and insectto DHICA DCDT and converts insect dopachrome DCDT converts to DHI dopachrome via a common to quinoneDHI via methide a common intermediate. quinone Dihydroxyindolesmethide intermediate. are oxidizedDihydroxyindoles to indolequinone, are oxid quinoneized to methideindolequinone, and iminochrome quinone methide isomers thatand williminochrome then polymerize isomers producing that will DHIthen melanin,polymerize DHICA producing melanin DHI and melanin, mixed DHI-DHICADHICA melanin melanin. and Themixed generation DHI-DHICA and subsequentmelanin. The reactions generation of semiquinones and subsequent will reaction also leads toof thesemiquinones melanin production will also andlead accountto the melanin for the freeproduction radical natureand account of melanin for the polymer. free radical nature of melanin polymer.

Figure 6.6.Modified Modified Raper–Mason Raper–Mason Pathway Pathway for the for Biosynthesis the Biosynthesis of Eumelanin. of Eumelanin. Tyrosinase (A)Tyrosinase catalyzes (A) the oxidationcatalyzes ofthe tyrosine oxidation to dopaquinone of tyrosine and to conversiondopaquinone of dopa and to conversion dopaquinone. of Dopaquinonedopa to dopaquinone. undergoes intermolecularDopaquinone undergoes cyclization intermolecular producing leucochrome, cyclization which producing undergoes leucochrome, double decomposition which undergoes with dopaquinonedouble decomposition generating with dopa dopaquinone and dopachrome. generating Dopachrome dopa isand isomerized dopachrome. by DCT Dopachrome to the transient is quinoneisomerized methide by DCT intermediate to the transi thatent is quinone converted methide to DHICA intermediate in mammals. that is Insect converted DCDT to on DHICA the other in handmammals. produces Insect DHI. DCDT Nonenzymatic on the other transformations hand produces mayDHI. also Nonenzymatic be responsible transformations for DHI versus may DHICA also productionbe responsible in differentfor DHI versus systems. DHICA Oxidative production polymerization in different of dihydroxyindolessystems. Oxidative generates polymerization different of kindsdihydroxyindoles of eumelanin generates pigments. differe (B =nt nonenzymatic kinds of eumelanin reactions; pigments. C = mammalian (B = nonenzymatic DCT; C0 = reactions; insect DCDT; C = andmammalian D = DHICA DCT; oxidase). C′ = insect DCDT; and D = DHICA oxidase).

The bulk of the studies carried out in relation to Raper–Mason pathway indicate that DHI is the The bulk of the studies carried out in relation to Raper–Mason pathway indicate that DHI is major product of dopachrome conversion while DHICA is a minor product [10–14]. However, the major product of dopachrome conversion while DHICA is a minor product [10–14]. However, mammalian DCT produces only DHICA and not DHI [18–20,33–35]. Moreover, DCT of mammalian mammalian DCT produces only DHICA and not DHI [18–20,33–35]. Moreover, DCT of mammalian origin seems to be associated with the tyrosinase as high molecular weight complex of tyrosinase origin seems to be associated with the tyrosinase as high molecular weight complex of tyrosinase and and related proteins [17]. Such a metabolon complex will channel the product of one reaction into related proteins [17]. Such a metabolon complex will channel the product of one reaction into the active the active site of the other enzyme to ensure an efficient metabolic transformation. Complex site of the other enzyme to ensure an efficient metabolic transformation. Complex formation also avoids formation also avoids the leakage of harmful quinonoid reactive intermediates. This constraint the leakage of harmful quinonoid reactive intermediates. This constraint dictates the sole production dictates the sole production of DHICA melanin and not DHI melanin. However, all three different of DHICA melanin and not DHI melanin. However, all three different eumelanins, viz., DHI melanin, eumelanins, viz., DHI melanin, DHICA-DHI mixed melanin and DHICA melanin, are formed in DHICA-DHI mixed melanin and DHICA melanin, are formed in mammalian systems [3,5,42,43]. mammalian systems [3,5,42,43]. For this to occur, obviously DHI has to be synthesized some how in For this to occur, obviously DHI has to be synthesized some how in comparable quantities to that of comparable quantities to that of DHICA or even more. It is possible that there is an undiscovered DHICA or even more. It is possible that there is an undiscovered dopachrome-converting enzyme that dopachrome-converting enzyme that specifically converts dopachrome to DHI in mammals, or nonenzymatic reactions are playing a crucial role to generate DHI in different melanogenic systems. It should be recalled here that various divalent metal ions are assisting the production of only

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Int. J. Mol. Sci. 2016, 17, 1753 8 of 24 specifically converts dopachrome to DHI in mammals, or nonenzymatic reactions are playing a crucial roleDHICA to generate in nonenzymatic DHI in different reactions melanogenic [25]. Thus systems. the conditions It should for be recalledDHI production here that variousin melanogenic divalent metalsystem ions have are to assisting be devoid the production of bivalent of onlymetal DHICA ions to in ensure nonenzymatic nonenzymatic reactions decarboxylative [25]. Thus the conditionstransformation for DHIof dopachrome production to in DHI. melanogenic Certainly system more studies have to are be needed devoid to of clarify bivalent the metal biosynthetic ions to ensureorigin of nonenzymatic DHI in mammalian decarboxylative systems. Similarly, transformation in insects of dopachrome system the topresence DHI. Certainly or absence more of DHICA studies aremelanin needed should to clarify be unequivocally the biosynthetic assessed. origin of DHI in mammalian systems. Similarly, in insects system the presence or absence of DHICA melanin should be unequivocally assessed. 9. Melanin from Other Catecholamines 9. Melanin from Other Catecholamines Tyrosinase as well as phenoloxidases exhibit wide substrate specificity and oxidize a number of catecholicTyrosinase compounds. as well asIt phenoloxidasesis expected that exhibit not wideonly substratedopa but specificityalso a variety and oxidize of endogenous a number ofcatecholamines catecholic compounds. that can serve It isas expectedsubstrates that for the not oxidative only dopa enzymes, but also will a variety participate of endogenous in melanin catecholaminesbiosynthesis. Oxidative that can enzymes serve as also substrates attack fordopamine, the oxidative a very enzymes, common willmetabolite participate of dopa in melanin formed biosynthesis.by the action Oxidativeof dopa decarboxylase, enzymes also attackto dopa dopamine,minequinone. a very Dopaminequinone common metabolite exhibits of dopa the formed same bykind the of action intramolecular of dopa decarboxylase, cyclization and to dopaminequinone.oxidation producing Dopaminequinone dopaminechrome. exhibits Conversion the same of kinddopaminechrome of intramolecular to DHI cyclization and its further and oxidation oxidation will producing result in dopaminechrome.DHI melanin [44,45]. Conversion The course of dopaminechromethe reactions leading to DHIto the and generation its further of dopamine oxidation willmelanin result is indepicted DHI melanin in Figure [44 7.,45 As]. Theis evident course the of thepathway reactions is very leading similar to theto the generation one proposed of dopamine for dopa melanin melanin is with depicted less simple in Figure reactions7. As isdue evident to the thelack pathway of carboxyl is very group. similar What to theis oneintriguing proposed in this for dopapathway melanin is the with fact less that simple dopaminechrome reactions due toisomerization the lack of carboxylcan proceed group. without What the is intriguing need for inan this enzyme pathway [44,45], is the although fact that some dopaminechrome of the insect isomerizationDCDTs have canbeen proceed shown without to thecatalyze need forthe an conversion enzyme [44 ,45of], althoughdopaminechrome some of the to insect DHI DCDTs [46]. haveDopamine-melanin been shown to seems catalyze to be the the conversion dominant of form dopaminechrome of neuromelanin to DHI in substantia [46]. Dopamine-melanin nigra [47]. It is seemslikely that to be dopamine the dominant and form other of catecholamines neuromelanin in found substantia in this nigra tissue [47 ].are It isreadily likely thatoxidized dopamine either and by othermetal catecholaminesions or by other found enzymes in this to tissuereactive are quinon readilyoid oxidized species, either which by eventually metal ions orpolymerize by other enzymes to black tocolored reactive neuromelanin quinonoid species,[47]. Insects which also eventually seem to produce polymerize dopamine to black derived colored melanin neuromelanin much more [47]. Insectsthan dopa also derived seem to producemelanin dopamine(see Sections derived 11–13). melanin DHI can much also more react than with dopa molecular derived melaninoxygen (seeand Sectionsundergo 11 semiquinone–13). DHI can formation, also react withwhich molecular will result oxygen in radical and undergo coupling semiquinone reactions formation,leading to which DHI willmelanin. result The in radical structure coupling as reactionswell as leadingmechanism to DHI of melanin.formation The of structure neuromelanin as well as is mechanism still under of formationinvestigation of neuromelaninin several laboratories. is still under investigation in several laboratories.

Figure 7. MelaninMelanin biosynthesis biosynthesis from from do dopamine.pamine. Tyrosinase Tyrosinase or orother other oxidative oxidative enzymes/reactants enzymes/reactants will willconvert convert dopamine dopamine to dopaminequ to dopaminequinone.inone. Intramolecular Intramolecular cyclizat cyclizationion to to leucochrome leucochrome and and further oxidation of the leucochrome produces the dopaminechrome.dopaminechrome. Dopaminechrome is converted to DHI by isomerization reaction. Further Further oxidation oxidation of of DH DHII to its quinonoid products and their eventual polymerization leads to dopamine melanins,melanins, whichwhich isis DHIDHI melanin.melanin.

Norepinephrine (also known as noradrenaline) andand its methylated derivative, epinephrine (also called adrenaline) can also form melanin pigmen pigment.t. They They arise arise by by the the action action of of dopamine- dopamine-ββ-hydroxylase,-hydroxylase, which converts dopamine to norepinephrine. Nore Norepinephrinepinephrine is issubjecte subjectedd to tomethylation methylation by by a N-methyl transferase that adds a methyl group on to amine generating the catecholamine hormone—epinephrine. Both these catecholamines can be oxidized by tyrosinase and related oxidases to their corresponding quinones. These quinones will also exhibit intermolecular

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aInt.N J.-methyl Mol. Sci. 2016 transferase, 17, 1753 that adds a methyl group on to amine generating the catecholamine9 of 24 hormone—epinephrine. Both these catecholamines can be oxidized by tyrosinase and related oxidasescyclization to theirreaction corresponding much like quinones. dopaquinone These quinonesand dopaminequinone will also exhibit producing intermolecular leucochromes. cyclization reactionFurther oxidation much like of dopaquinone leucochrome and and dopaminequinone conversion to the producing transient leucochromes. quinone methide Further tautomer oxidation leads of leucochrometo a reaction and that conversion generates to thea keto transient catechol quinone [48, methide49]. In tautomerthe case leadsof adrenaline to a reaction oxidation, that generates this acompound keto catechol called [48, 49adrenolutin]. In the case has of adrenalinebeen isolated oxidation, and thischaracterized compound very called well adrenolutin [48]. A has similar been isolatedcompound and may characterized be formed very from well norepinephrine. [48]. A similar compoundEnolization may of the be formedketocatechol from norepinephrine.and its further Enolizationoxidation generates of the ketocatechol again polymeric and its melanin further oxidationpigments generates (Figure 8). again In mammalian polymeric melanin substantia pigments nigra, (Figuresuch melanin8). In mammalian formation substantiais possible. nigra, However, such melaninit is not formationclear if any is possible.such melanin However, is produced it is not clearfrom ifepinephrine any such melanin and norepinephrine is produced from in any epinephrine insect tissues. and norepinephrine in any insect tissues.

Figure 8.8. MelaninMelanin biosynthesis biosynthesis from epinephrine andand norepinephrine.norepinephrine. Both Both epinephrine and norepinephrine can can be be easily easily oxidized oxidized to their to two-electrontheir two-electron oxidation oxidation product—quinones. product—quinones. These quinones These alsoquinones exhibit also rapid exhibit intramolecular rapid intr cyclizationamolecular producing cyclization leucochromes. producing leucochromes. Conversion of leucochromeConversion toof iminochromeleucochrome to and iminochrome isomerization and to quinoneisomerization methide to will quinone result inmethide adrenolutin will result formation in adrenolutin in the case offormation epinephrine. in the A case similar of reactionepinephrine. of norepinephrine A similar reaction to the ketoof norepinephrine indole derivative to hasthe notketo yet indole been demonstrated,derivative has not but yet is quite been possible. demonstrated, Further but oxidative is quitetransformations possible. Further of oxidative these products transformations will result inof melaninthese products production. will result in melanin production.

Finally, there is one possible minor pathway for melanoid pigment production from a Finally, there is one possible minor pathway for melanoid pigment production from a derivative derivative of dopamine. 3,4-Dihydroxyphenylacetic acid is produced in a number of biological of dopamine. 3,4-Dihydroxyphenylacetic acid is produced in a number of biological systems systems as the degradation product of dopamine. Being an o-diphenol, it is also easily oxidized as the degradation product of dopamine. Being an o-diphenol, it is also easily oxidized practically by all tyrosinases and phenoloxidases. The quinone product exhibits three different practically by all tyrosinases and phenoloxidases. The quinone product exhibits three different reactions of which intramolecular cyclization parallels dopamine melanin formation [50]. Its rapid reactions of which intramolecular cyclization parallels dopamine melanin formation [50]. Its rapid intramolecular cyclization generates 2,5,6-trihydroxybenzofuran. This reactive trihydric phenol is intramolecular cyclization generates 2,5,6-trihydroxybenzofuran. This reactive trihydric phenol is further oxidized to furanoquinone and gets converted to a transient quinone methide (Figure 9). further oxidized to furanoquinone and gets converted to a transient quinone methide (Figure9). Eventually all these reactive compounds polymerize and produce a black colored insoluble Eventually all these reactive compounds polymerize and produce a black colored insoluble melanoid melanoid pigment [50]. 3,4-Dihydroxyphenylacetic acid is produced as a degradation product of pigment [50]. 3,4-Dihydroxyphenylacetic acid is produced as a degradation product of dopamine dopamine in mammalian as well as insect systems. However, whether the melanoid product derived in mammalian as well as insect systems. However, whether the melanoid product derived from 3,4-dihydroxyphenylacetic acid is formed in any biological system or not remains to be from 3,4-dihydroxyphenylacetic acid is formed in any biological system or not remains to be determined. Certainly more work is needed on this and other catecholamine derivatives destined for determined. Certainly more work is needed on this and other catecholamine derivatives destined for oxidative polymerization. oxidative polymerization.

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Figure 9. Oxidative cyclization of 3,4-dihydroxyphenylacetic acid. The quinone formed by the Figure 9. Oxidative cyclization of 3,4-dihydroxyphenylacetic acid. The quinone formed by the oxidation oxidation of 3,4-Dihydroxyphenylacetic acid among other reactions exhibits an intramolecular of 3,4-Dihydroxyphenylacetic acid among other reactions exhibits an intramolecular cyclization and cyclization and produces a trihydroxybenzofuran, which undergoes oxidative polymerization producesFigure a trihydroxybenzofuran,9. Oxidative cyclization whichof 3,4-dihydroxyphe undergoes oxidativenylacetic polymerization acid. The quinone yielding formed a black by coloredthe oxidationyielding a blackof 3,4-Dihydroxyphenylacetic colored melanoid pigment. acid among other reactions exhibits an intramolecular melanoid pigment. cyclization and produces a trihydroxybenzofuran, which undergoes oxidative polymerization 10. Pheomelaninyielding a black colored melanoid pigment. 10. Pheomelanin Pheomelanin is a branch point in the overall melanin biosynthetic process [2,3,5,8,9,42]. 10.DopaquinonePheomelanin Pheomelanin as is well a branchas dopamine point quinone in the that overall are formed melanin by biosynthetic the action of processtyrosinase [2 ,exhibit3,5,8,9 ,42]. DopaquinoneinstantaneousPheomelanin as nonenzymatic well is asa dopaminebranch addition point quinone reactionsin the thatoverall with are endogenous melanin formed biosynthetic by thiols the such action asprocess cysteine of tyrosinase [2,3,5,8,9,42]. producing exhibit instantaneousDopaquinonethiolated catecholamines. nonenzymatic as well as dopamineThese addition thiolated quinone reactions catecholam that with are endogenousines formed are the by buildingthe thiols action such blocks of as tyrosinase cysteineof pheomelanin. producingexhibit thiolatedinstantaneousThe availability catecholamines. nonenzymatic of thiols Thesein any addition system thiolated reactions will catecholamines dictate with how endogenous much are pheomelanin the thiols building such will as blocks cysteinebe synthesized of producing pheomelanin. in a Thethiolatedgiven availability tissue. catecholamines. ofTissues thiols rich in anyThese in reduced system thiolated willglutathione catecholam dictate howorines cysteinemuch are the pheomelaninnaturally building fa blocksvor will the of be productionpheomelanin. synthesized of in thiolated catecholamine derivatives and hence pheomelanin. Tissues poor in thiols will not be able a givenThe availability tissue. Tissues of thiols rich in inany reduced system will glutathione dictate how or much cysteine pheomelanin naturally will favor be synthesized the production in a of givento synthesize tissue. Tissuessignificant rich amounts in reduced of pheomelanin. glutathione orThiols cysteine add naturallyonto quinones favor bythe a productionquite strange of thiolated catecholamine derivatives and hence pheomelanin. Tissues poor in thiols will not be able to thiolatedreaction andcatecholamine do not follow derivatives the typical and hence nucleophilic pheomelanin. addition Tissues rules. poor Thiols in thiols produce will not uniquely be able synthesize significant amounts of pheomelanin. Thiols add onto quinones by a quite strange reaction to1,6-addition synthesize products significant rather amounts than 1,4-addition of pheomelanin. products, Thiols as shown add onto in Figure quinones 10 [42,51–53]. by a quite strange andreaction do not followand do thenot typical follow nucleophilicthe typical nucleophilic addition rules. addition Thiols rules. produce Thiols uniquelyproduce 1,6-additionuniquely products1,6-addition rather products than 1,4-addition rather than products, 1,4-addition as shownproducts, in Figureas shown 10 in[42 Figure,51–53 10]. [42,51–53].

Figure 10. Thiol addition to dopaquinone. Thiols such as cysteine add on to dopaquinone not by conventional Michael-1,4-addition reaction. They react by an unconventional 1,6-addition, producing 5-S-cysteinyl dopa as the major product and 2-S-cysteinyldopa as the minor product. In FigureFigure 10. 10.Thiol Thiol addition addition to to dopaquinone. dopaquinone. Thiols such such as as cysteine cysteine add add on on to todopaquinone dopaquinone not notby by addition 2,5-dicysteinyldopa is also formed to a certain extent. RSH—Cysteine. conventionalconventional Michael-1,4-addition Michael-1,4-addition reaction. reaction. They Th reactey react by an by unconventional an unconventional 1,6-addition, 1,6-addition, producing producing 5-S-cysteinyl dopa as the major product and 2-S-cysteinyldopa as the minor product. In 5-S-cysteinyl dopa as the major product and 2-S-cysteinyldopa as the minor product. In addition additionThe thiolated 2,5-dicysteinyldop catechols area is further also formed oxidized to a certain to their extent. corresponding RSH—Cysteine. quinones most likely by the endogenous2,5-dicysteinyldopa dopaquinone. is also formedThe cysteinyl to a certain amino extent. group RSH—Cysteine. is situated in a suitable position to form a cyclicThe adduct. thiolated This catechols reaction givesare further rise to oxidized a quinone to iminetheir corresponding that could either quinones undergo most decarboxylative likely by the endogenous The thiolated dopaquinone. catechols areThe furthercysteinyl oxidized amino group to their is situated corresponding in a suitable quinones position most to form likely a by the endogenouscyclic adduct. dopaquinone.This reaction gives The rise cysteinyl to a quinone amino imine group that is could situated either in undergo a suitable decarboxylative position to form

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Int. J. Mol. Sci. 2016, 17, 1753 11 of 24 a cyclic adduct. This reaction gives rise to a quinone imine that could either undergo decarboxylative aromatizationaromatization or or simple simple isomerizationisomerization coupled coupled ar aromatizationomatization producing producing the thebenzothiazine benzothiazine and its and its3-carboxylic 3-carboxylic derivative derivative respectively respectively [2,3,5,8,9 [2,3,5,8,,42,51–53].9,42,51–53 ].Oxidative Oxidative polymerization polymerization of ofthese these two two derivativesderivatives leads leads to to the the majority majority ofof thethe redred toto brownbrown colored pheomelanin pheomelanin pigments pigments observed observed in in animalsanimals (Figure (Figure 11 11).).

FigureFigure 11. 11. AA simplifiedsimplified modelmodel forfor thethe biosynthesisbiosynthesis of pheomelanin.pheomelanin. Using Using the the major major product—5-product—5-S-cysteinyldopa—asS-cysteinyldopa—as the the precursor, precursor, the the biosynthesis biosynthes ofis pheomelanin of pheomelanin is illustrated is illustrated in this in this figure. figure. Oxidation of 5-S-cysteinyldopa produces its quinone, which undergoes internal condensation Oxidation of 5-S-cysteinyldopa produces its quinone, which undergoes internal condensation reaction reaction producing the quinone imine. Quinone imine either rearranges to form producing the quinone imine. Quinone imine either rearranges to form benzothiazine-3-carboxylic acid benzothiazine-3-carboxylic acid or undergoes decarboxylation to benzothiazine. Oxidative or undergoes decarboxylation to benzothiazine. Oxidative polymerization of these two compounds polymerization of these two compounds produces the majority of the red to brown pheomelanin produces the majority of the red to brown pheomelanin pigment in all animals. pigment in all animals.

MammalianMammalian systems systems produce produce significant significant amounts amounts of pheomelaninof pheomelani [42n, 43[42,43].]. In contrast, In contrast, the reports the onreports the presence on the presence of pheomelanin of pheomelanin in invertebrate in inverteb animalsrate animals and especiallyand especially insects insects are are very very few few [54 ]. Insects[54]. withInsects their with poor their reserves poor reserves for thiols for may thiols produce may produce much lessmuch pheomelanin. less pheomelanin. Insects Insects make melaninmake formelanin a variety for of a reasonsvariety of and reasons it is extremely and it is extremel importanty important for their for successful their successful survival. survival. However, However, studies aimedstudies at differentiatingaimed at differentiating between eumelaninbetween eumela andnin pheomelanin and pheomelanin in insects in areinsects few are and few far and between. far Firstbetween. proposal First that proposal pheomelanin that pheome couldlanin be could formed be informed insects in wasinsects pointed was pointed out by out Latocha by Latocha et al. et [55 ]. Theseal. [55]. authors These performed authors performed pyrolytic pyrolytic gas chromatography—mass gas chromatography—mass spectrometry spectrometry analysis analysis of melanin of frommelanin black, from gray black, and yellow gray and colored yellowDrosophila colored melanogasterDrosophila melanogasterand found theand presencefound the of presence pheomelanin of relatedpheomelanin compounds related in thecompounds yellow strain. in the yellow More recently, strain. More Galván recently, et al. Galván [54] provided et al. [54] the provided first concrete the first concrete evidence for the presence of pheomelanin in insects. They performed Raman evidence for the presence of pheomelanin in insects. They performed Raman spectroscopic studies on spectroscopic studies on the grasshopper and revealed the presence of eumelanin in black regions of the grasshopper and revealed the presence of eumelanin in black regions of cuticle and pheomelanin cuticle and pheomelanin in the yellowish red region of the cuticle. Moreover, HPLC analysis of the in the yellowish red region of the cuticle. Moreover, HPLC analysis of the degradation products degradation products from cuticular melanin revealed the presence of cysteinyldopamine derived from cuticular melanin revealed the presence of cysteinyldopamine derived pheomelanin products pheomelanin products thereby confirming the production of pheomelanin in insects. Pheomelanin thereby confirming the production of pheomelanin in insects. Pheomelanin has also been identified has also been identified recently in parasitic wasps [56]. The vertebrate pheomelanin found in the recentlyperipheral in parasitic tissues are wasps usually [56 made]. The up vertebrate of cysteinyldopa pheomelanin units [54], found where in as the the peripheral neuromelanin tissues may are usuallycontain made both up dopa of cysteinyldopa and dopamine units derived [54], wherepheomela as thenins neuromelanin [54]. Interestingly, may containinsect pheomelanin both dopa and dopamineseems to derived be derived pheomelanins mostly from [54]. cysteinyldopamine Interestingly, insect rather pheomelanin than cysteinyldopa. seems to be derivedIt would mostly be frominteresting cysteinyldopamine to pursue the rather role of thanpheomelanins cysteinyldopa. in insects It wouldespecially be in interesting the host-defense to pursue mechanisms the role of pheomelaninsand cuticular in hardening insects especially reactions. in Certainly, the host-defense more studies mechanisms are needed and to cuticular shed light hardening on the presence, reactions. Certainly,physiological more studiesrole and are biosynthesis needed to of shed pheomelanin light on the in presence,insects. physiological role and biosynthesis of pheomelanin in insects. 11. Insects Use Melanization and Sclerotization for Cuticular Coloration 11. Insects Use Melanization and Sclerotization for Cuticular Coloration In insects, a process that is very much similar to melanization occurs simultaneously and independently.In insects, a This process process that called is very sclerotization, much similar is extremely to melanization vital for the occurs survival simultaneously of most insects. andIt independently.aids the hardening This processof their calledexoskeleton sclerotization, (=cuticle) isan extremelyd protects vitalthe soft for bodies the survival at various of most stages insects. of

Int. J. Mol. Sci. 2016, 17, 1753 12 of 24

insect’s life [57–61]. Unhardened cuticle allows dehydration and desiccation of the insect, and is susceptible to easy invasion by foreign organisms [57–61]. Hardened cuticle apart from protecting the insects against external harsh elements serves as a point for attachment of muscles and other organs. Therefore hardening of the cuticle is an indispensable process for practically all insects. Our laboratory has been investigating the molecular mechanism of sclerotization for over three decades and has clarified the molecular mechanisms of cuticular sclerotization, which is summarized in Figure 12 [4,57–59]. Int. J. Mol. Sci.At 2016the ,beginning17, 1753 of sclerotization, cuticular phenoloxidases oxidize catecholamine derivatives12 of 24 such as N-acetyldopamine (NADA) and N-β-alanyldopamine (NBAD) to their corresponding quinones. These quinones may act as gluing agents and bond to cuticular components most notably, chitin and It aidsstructural the hardening proteins of[57–61], their exoskeletonor serve as substrates (=cuticle) for and the protectsnext enzyme the softin the bodies metabolic at various pathway stages of insect’scalled lifequinone [57– 61isomerase]. Unhardened [62–64]. cuticleQuinone allows isomerase dehydration converts 4-alkylquinones and desiccation to ofhydroxyquinone the insect, and is susceptiblemethides to and easy provide invasion them by foreignfor: (a) organismsquinone me [thide57–61 tanning]. Hardened wherecuticle in the apartside chain from of protecting the the insectscatecholamine against forms external adducts harsh with elements the regeneration serves of as the a pointcatecholamine for attachment ring; or (b) of the muscles next enzyme and other in the pathway—quinone methide isomerase [63,65,66]. Quinone methide isomerase acts on unstable organs. Therefore hardening of the cuticle is an indispensable process for practically all insects. NADA and NBAD quinone methides and converts them to 1,2-dehydro-N-acyldopamine derivatives Our laboratory[63,65,66]. Further has been oxidation investigating of dehydro the dopami molecularne derivatives mechanism produces of sclerotization the reactive for quinone over three decadesmethide and hasimine clarified amides the that molecular crosslink mechanismsproteins and ofchitin cuticular or react sclerotization, with parent catechols which is summarizedforming in Figuredimers, 12[ trimers4,57–59 and]. other oligomers [67–69].

Figure 12. Unified mechanism for sclerotization of insect cuticle. Sclerotizing precursors such as Figure 12. Unified mechanism for sclerotization of insect cuticle. Sclerotizing precursors such N-acetyldopamine (R = CH3) and N-β-alanyldopamine (R = CH2CH2NH2) are oxidized by cuticular as N-acetyldopamine (R = CH ) and N-β-alanyldopamine (R = CH CH NH ) are oxidized by phenoloxidases (A) to their corre3 sponding quinones, which participate in2 quinone2 tanning2 reaction cuticular phenoloxidases (A) to their corresponding quinones, which participate in quinone tanning by forming Michael-1,4-addition reaction with cuticular nucleophiles. Quinone isomerase (B) reactionconverts by forming part of the Michael-1,4-addition quinones to quinone methides reaction and with provide cuticular for quinone nucleophiles. methide Quinonetanning through isomerase (B) convertsMichael-1,6-addition part of the reactions. quinones Some to quinone of the quin methidesone methide and providealso serves for as quinone substrate methide for quinone tanning throughmethide Michael-1,6-addition isomerase (C), which reactions. transforms Some them of theto 1,2-dehydro- quinone methideN-acyldopamines. also serves Oxidation as substrate of for quinonedehydro methide compounds isomerase by (C),phenoloxidase which transforms generates them the tobifunctional 1,2-dehydro- quinoneN-acyldopamines. methide imine Oxidationamides of dehydrothat compoundsform adducts by with phenoloxidase both the side generates chain carbon the bifunctionalatoms. Somequinone of the dehydro methide compound imine amides also that formundergoes adducts with oligomerization both the side reaction chain (D carbon = nonenzymatic atoms. Some reactions). of the dehydro compound also undergoes oligomerization reaction (D = nonenzymatic reactions). In many insects, sclerotization is associated with coloration of cuticle, which is either due to melanin or due to quinonoid end products of sclerotizing catechols or a combination of both [59,60]. AtCuticles the beginning using NBAD of sclerotization,are often brown cuticularcolored, which phenoloxidases may have both oxidize melanin catecholamine and colored sclerotin derivatives such asendN products-acetyldopamine [59,60]. On (NADA) the other and hand,N-β cuticles-alanyldopamine using NADA (NBAD) are often to colorless their corresponding but are hardened quinones. Theseby quinones sclerotization may reactions act as gluing [60]. Both agents sclerotizati and bondon toand cuticular melanization components are two different most notably, biochemical chitin and structuralprocesses proteins with [different57–61], orphysiological serve as substrates roles and forare theused next for enzymeentirely different in the metabolic purposes. pathway However, called quinonethere isomerase exist many [62 similarities–64]. Quinone in these isomerase two processe convertss [57]. 4-alkylquinonesBoth processes are to initiated hydroxyquinone by the enzyme, methides and providephenoloxidase them for:or tyrosina (a) quinonese, which methide oxidizes tanning the catecholic where in precursors the side chainto their of thecorresponding catecholamine forms adducts with the regeneration of the catecholamine ring; or (b) the next enzyme in the pathway—quinone methide isomerase [63,65,66]. Quinone methide isomerase acts on unstable NADA and NBAD quinone methides and converts them to 1,2-dehydro-N-acyldopamine derivatives [63,65,66]. Further oxidation of dehydro dopamine derivatives produces the reactive quinone methide imine amides that crosslink proteins and chitin or react with parent catechols forming dimers, trimers and other oligomers [67–69]. In many insects, sclerotization is associated with coloration of cuticle, which is either due to melanin or due to quinonoid end products of sclerotizing catechols or a combination of both [59,60]. Cuticles using NBAD are often brown colored, which may have both melanin and colored sclerotin end products [59,60]. On the other hand, cuticles using NADA are often colorless but are hardened by sclerotization reactions [60]. Both sclerotization and melanization are two different biochemical processes with different physiological roles and are used for entirely different purposes. However, Int. J. Mol. Sci. 2016, 17, 1753 13 of 24 there exist many similarities in these two processes [57]. Both processes are initiated by the enzyme, phenoloxidase or tyrosinase, which oxidizes the catecholic precursors to their corresponding quinones. In sclerotization, the quinone cannot undergo intramolecular nucleophilic addition reaction as the amino group is protected by acylation. Therefore the reaction is diverted to external nucleophiles. However, in melanogenic process, the quinone exhibits intramolecular cyclization due to the presence of appropriately positioned amine group. The next set of reactions to be considered is the transformation of quinone to quinone methide. In sclerotization, quinone isomerase performs the conversion of N-acyldopamine quinones to quinone methides [62–64]. The parallel reaction in melanogenesis is catalyzed by DCT that converts dopachrome to quinone methide [57]. The next reaction in sclerotization is the conversion of quinone methide to 1,2-dehydro-N-acyldopamine catalyzed by the enzyme, quinone methide isomerase [63,65,66]. The similar reaction in melanogenesis may be assisted by DCT itself, or occur via nonenzymatic conversion as the driving force for such a reaction could come from the aromatization of quinonoid nucleus to benzenoid structure [57]. In both cases, the side chain of the catecholamine is specifically dehydrogenated to produce a desaturated compound. Generation of side chain desaturated compound is absolutely essential for both the processes as it provides additional loci for adduct and/or crosslink formation. Towards the end, the oxidation of desaturated catechols to its quinonoid products takes place. In sclerotization, phenoloxidase or even nonenzymatic reactions could aid the oxidation process. In melanization DHICA oxidation is aided by DHICA oxidase, while DHI oxidation may be tyrosinase catalyzed or nonenzymatic in nature [36–38]. These oxidations produce in both cases, oligomeric products before polymers are generated. In the case of dehydro NADA, we have demonstrated the production of not only dimers and trimers, but also oligomers up to hexamers by mass spectral studies [69]. Similarly, for both DHICA and DHI, oligomeric products have been characterized [70–74]. In spite of such remarkable similarities, significant differences exist between melanin biosynthesis and sclerotization reactions [4,57–59]. The reactivity of the catecholamines in melanin biosynthesis is mostly confined to internal cyclization and self-polymerization, although nucleophiles from proteins and other molecules are also incorporated at later stages. However the reactivity of catecholamine derivatives in sclerotization are mostly directed to external reactivities with other nucleophiles with the exception of some oligomerization reaction. In other words, catecholamines are used to glue and bond structural proteins and chitin polymers together to form a supramolecular structure in the case of cuticular sclerotization [57–61]. On the other hand, in melanin biosynthesis, the majority of the catecholamines are used for the production of polymeric melanin pigment [2,3,5,9,42]. Thus the quinone products formed in sclerotization are destined for external reactivities, while the quinones generated during melanogenesis are mostly used for internal reactivity with the exception of pheomelanogenesis. In this instance, dopaquinone and related compounds form adducts with external sulfur nucleophiles forming thiolated catechols. Finally, a note on the enzymes associated with the initiation of these two processes. The insect tyrosinases do not have any significant homology with the mammalian tyrosinases [75]. In insects, the sclerotization process is initiated by laccase, which is a p-diphenoloxidase rather than tyrosinase, which is a typical o-diphenoloxidase [76]. Laccases oxidize both o-diphenols and p-diphenols. Tyrosinases do not oxidize p-diphenols but do oxidize o-diphenols. Tyrosinase uses a two-electron oxidation process to generate the quinone directly from their catecholic substrates. Laccases on the other hand, use one-electron oxidation producing semiquinone as the primary products [77]. The semiquinone, however, undergoes disproportionation reaction to form the parent diphenol and quinone product. Thus, both tyrosinases and laccases, which are structurally unrelated to each other, generate the same quinonoid product from their diphenolic substrates. There are major differences in melanin biosynthesis that occurs in mammalian system and insect systems [4,57]. Melanin biosynthesis in mammals is limited mostly to specialized cells called melanocytes [2]. All the enzymes associated with melanin biosynthesis are confined to this organelle. Often these enzymes are membrane bound, inextractable and form a tight complex with one another and with other melanogenic related proteins [17]. In insects, melanin is made both in the exoskeleton Int. J. Mol. Sci. 2016, 17, 1753 14 of 24 and inside the body. The cuticular phenoloxidases tend to be cuticle-bound enzymes while, hemolymph phenoloxidases are of soluble type. The enzymes associated with insect melanogenesis are present in their hemolymph (=blood), which bathes the entire insect body and hence these enzymes are distributed through out the insect body [4,57]. Tyrosinase is present in its active form in melanocytes. However, having the active phenoloxidase in the insect hemolymph is very deleterious to the insects, as its substrates are also present in the circulating blood and hence they are subjected to oxidation [4,57]. This interaction can result in the production of undesired quinonoid toxic compounds. Hence insects keep the phenoloxidase in the inactive proenzyme form and activate it during a specific need. As stated early, mammalian tyrosinase performs both the oxidation of tyrosine and dopa to dopaquinone. However, insect phenoloxidases do not seem to possess much of “tyrosine hydroxylase” activity. Insects mostly use specific tyrosine hydroxylase for dopa production. There are significant differences at the stage of dopachrome conversion reaction also [4,57]. Mammalian DCT converts dopachrome to DHICA [18–20]. However, insect DCDT that were characterized so far, only produce DHI as the sole product [4,21–23,46,57]. Whether nonenzymatically dopachrome is converted to DHICA or not in insects is yet to be assessed. As a consequence, insects may be specifically making DHI melanin [78] and not the DHICA or mixed melanin as is observed in mammalian melanogenic process. Eumelanin thus seems to be the dominant melanin present in the insects [54]. Even though the presence of pheomelanin in three insect systems has been reported, its role and abundance in insect is yet to be determined [54–56].

12. Importance of Melanin for Insect Biochemistry and Physiology Insects use melanin biosynthesis for a variety of physiologically important processes that are extremely vital for their survival [4,57–61,79,80]. Cuticular melanization is one of the important processes. As stated earlier, both sclerotization and melanization often occur in the same cuticle to give different color and strength to the cuticle. Insects have developed an amazing array of coloring pattern for their body to help them conceal against specific backgrounds in different environments for better survival. The coloring patterns developed over a period of several generations by natural selection process have helped them to avoid predation. During industrial revolution, pepper moths evolved darker coloration of cuticle to avoid predation on trees coated with darker industrial pollutants. This process referred to as industrial melanism is a classical case for evolution of color variation [79,80]. Another physiological process where melanin plays an essential role is wound healing/sealing reactions [81–84]. During wounding, massive deposition of melanin occurs at the wound site in insects for two major reasons. One it helps to seal the wound along with other biochemical processes to prevent further blood loss. Second, melanogenic quinonoid products are well known cytotoxic compounds. Therefore, they will serve to kill any opportunistic microorganisms trying to enter the insect body through the wound site [81–84]. Melanin also plays a third important role in host defense reactions or innate immune responses [4,57–61,81–86]. Higher animals have evolved an elaborate and complex immune system that can exhibit both memory and specificity to recognize and respond to various infectious agents. For example, they use lymphocytes and specific antibodies to ward of infections successfully. Lacking such vital immune response components, insects have managed to defend themselves against invading pathogens, which gain access into the insect by a plethora of host defense reactions. Melanization is an important and indispensable component of this innate immune responses [4,57–61,81–86]. Insects use a wide range of cellular and humoral defense reactions to protect themselves. The cellular responses are phagocytosis, nodule formation and encapsulation, while the humeral responses are antibacterial peptide synthesis production, use of lectin type molecules and activation of prophenoloxidase cascade. Organisms entering the insects are often found encapsulated and melanized. Therefore, phenoloxidase and melanin are considered as important components of insect’s immune system. This process limits the damage caused by the intruder. Again, the cytotoxic quinonoid products formed during the process also help the insect to fight the invading organisms. A lot of Int. J. Mol. Sci. 2016, 17, 1753 15 of 24 review articles have appeared in the literature time to time on this topic [4,81–86]. The rest of the article will focus mainly on the cuticular pigmentation and melanization, as it has become an intensive area of research in recent years.

13. Insect Genes Associated with Melanization and Cuticular Sclerotization Int. J. Mol. Sci. 2016, 17, 1753 15 of 24 Several genes are associated with cuticular hardening and sclerotization pathways. However, 13. Insect Genes Associated with Melanization and Cuticular Sclerotization only some of the important genes that are directly involved in these two processes are shown Several genes are associated with cuticular hardening and sclerotization pathways. However, in the Figure 13 and only these will be discussed. Thus far, eight genes have been identified to only some of the important genes that are directly involved in these two processes are shown in the play a crucialFigure role13 and in insectonly these cuticular will be pigmentationdiscussed. Thus far, and eight sclerotization genes have been process. identified These to play are palea , DDC, aaNAT, ebonycrucial, tanrole, inblack insect, Laccase cuticular 2 ,pigmentation and yellow and(f and sclerotizationf2), which process. code These for tyrosine are pale, DDC hydroxylase,, aaNAT, dopa decarboxylase,ebony, tan arylalkylamine-, black, Laccase 2,N and-acetyltransferase, yellow (f and f2), NBADwhich code synthetase, for tyrosine NBAD hydroxylase, hydrolase, dopa aspartate decarboxylase,decarboxylase, laccase arylalkylamine- associated withN-acetyltransferase, cuticular sclerotization, NBAD synthetase, and DCDT, NBAD respectively.hydrolase, aspartate The genes for decarboxylase, laccase associated with cuticular sclerotization, and DCDT, respectively. The genes quinone isomerasefor quinone andisomerase quinone and quinone methide methide isomerase isomeras associatede associated with with cuticular cuticular sclerotization areare yet to be identified.yet to be identified.

Figure 13. Genes associated with melanization and sclerotization processes in insect cuticle. Insect Figure 13. Genes associated with melanization and sclerotization processes in insect cuticle. cuticular melanogenesis is intimately linked to cuticular sclerotization process, which makes the Insect cuticularcuticle strong melanogenesis and hard by iscrosslinking intimately cuticular linked proteins to cuticular and chitin. sclerotization Both processes process, start with which the makes the cuticleamino strong acid, and tyrosine. hard Tyrosine by crosslinking hydroxylase cuticularconverts tyrosine proteins to do andpa. Dopa chitin. decarboxylase Both processes generates start with the aminodopamine acid, tyrosine.from dopa. TyrosineBoth dopa hydroxylaseand dopamine can converts serve as tyrosinethe precursor to dopa.for melanin. Dopa Cuticular decarboxylase generateslaccase dopamine seems fromto be responsible dopa. Both for dopamelanization and dopamine in cuticle by can oxidizing serveas these the two precursor catechols for to melanin. quinones. Both these quinones after conversion to chromes serve as substrates for DCDT, which Cuticular laccase seems to be responsible for melanization in cuticle by oxidizing these two catechols to produce DHI as the product. Oxidative polymerization DHI makes the black colored melanin quinones.pigment Both these in cuticle. quinones Most importantly, after conversion dopamine to chromesis primarily serve converted as substrates to NBADfor and DCDT, NADA whichby the produce DHI as theaction product. of NBAD Oxidative synthetase polymerization and N-acetyl DHItransferase makes respectively the black. coloredOxidation melanin of thesepigment catechols in cuticle. Most importantly,produces their dopamine quinones, which is primarily are acted convertedon by quinone to isomerase NBADand and qu NADAinone methide by the isomerase action of NBAD synthetasesequentially and N-acetyl leading transferase to the generation respectively. of dehy Oxidationdro compounds. of these Oxidative catechols coupling produces of dehydro their quinones, compounds along with the adduct formation by N-acyldopamine quinones and quinone methides which areaccount acted for on sclerotization by quinone reactions. isomerase (Enzymes and quinone catalyzing methide various isomerasereactions are sequentially labeled in red. leading The to the generationgenes of dehydroassociated compounds.with these enzymes Oxidative are named coupling in blue. Black of dehydro arrow enzyme compounds catalyzed along reactions; with red the adduct formationarrow by N nonenzymatic-acyldopamine reactions). quinones and quinone methides account for sclerotization reactions. (Enzymes catalyzing various reactions are labeled in red. The genes associated with these enzymes are The hydroxylation of tyrosine to dopa is the first step involved in the biosynthesis of the named in blue. Black arrow enzyme catalyzed reactions; red arrow nonenzymatic reactions). catecholamines. This reaction is specifically catalyzed by tyrosine hydroxylase (TH) encoded by the pale gene [87]. Tyrosine hydroxylase is an iron containing monooxygenase incorporating one atom of The hydroxylation of tyrosine to dopa is the first step involved in the biosynthesis of the catecholamines. This reaction is specifically catalyzed by tyrosine hydroxylase (TH) encoded by Int. J. Mol. Sci. 2016, 17, 1753 16 of 24 the pale gene [87]. Tyrosine hydroxylase is an iron containing monooxygenase incorporating one atom of molecular oxygen into tyrosine and reducing the other atom to water with the help of tetrahydrobiopterin cofactor [88]. The enzyme contains an amino terminal hydrophilic regulatory domain that has several phosphorylation sites and a carboxy terminal hydrophobic domain, which has catalytic site and cofactor binding site [88]. In Drosophila alternative splicing of the primary transcript produces two isoforms TH1 and TH2, which are expressed in different tissues [87]. They also have different phosphorylation sites, and different kinetic parameters. TH1 is expressed in the central nervous system and is activated by phosphorylation. This isoform is mainly associated with the production of catecholamine derivatives distained for neurotransmission. Isozyme TH2 is expressed in epidermis and is responsible for the production of dopa needed for cuticular sclerotization and melanization [87]. Importantly, tyrosine hydroxylase is required for the survival of Drosophila as embryos that are null mutant of pale are unpigmented and fail to hatch. Similarly, its essential role for the survival of the silkworm, Bombyx mori has also been documented [89]. The DDC gene codes the next important enzyme, dopa decarboxylase. It is a pyridoxal-50-phosphate containing enzyme, playing a pivotal role in the biosynthesis of neurotransmitters, dopamine, and serotonin by specifically decarboxylating dopa and 5-hydroxytryptophan, respectively. Dopamine is absolutely essential for the biosynthesis of both melanin and sclerotin in insects [57–61,78–81]. Drosophila has five aromatic-decarboxylase genes of which, only one is responsible for the catalyzing the decarboxylation of dopa. Two DDC-like genes are characterized as α-methyl-dopa resistant proteins [90] and the two other are identified as tyrosine decarboxylase genes [91]. The catalytically active form of dopa decarboxylase exists as a homodimer and is expressed in the epidermis. At the active site, the enzyme has a H192, which is associated with pyridoxal phosphate binding and a Thr82 that is involved in substrate binding [92]. Loss of function at the DDC locus by mutation is often lethal. The cuticle of surviving flies is very soft and mostly unpigmented, a phenotype that emphasizes the vital need of dopamine for both sclerotization and melanization reactions. The aaNAT gene encodes arylalkylamine-N-acetyltransferases. In mammals, this protein catalyzes the essential first step in the formation of melatonin in the pineal gland and is associated with circadian rhythm. In insects, the aaNAT enzymes have different physiological roles [93,94]. They catalyze the formation of one of the two widely used insect cuticular sclerotizing precursors, NADA. They are also important for the inactivation of a number of biogenically produced monoamines (norepinephrine, dopamine, octopamine) due to the fact that insects do not possess monoamine oxidase for such inactivation. Acetyl CoA is the co-substrate for aaNAT in producing the N-acetyl derivatives of amines [95]. Recent studies point out that aaNAT suppresses melanin pigmentation in some insect cuticle [93–99]. The adult melanism (mln) mutant of silkworm Bombyx mori exhibits a strong black coloration in contrast to the wild type’s normal coloration [96]. Studies indicate that this black coloration correlates with loss of aaNAT function in mln gene. In the mln larvae, only the regions that are normally heavily sclerotized show an abnormal pigmentation. All these results point that aaNAT has an important role in normal coloration of cuticle and its loss of function by mutation causes possible build up of excess dopamine that is then diverted for dopamine melanin production [96,97]. NBAD synthetase coded by the ebony gene is expressed in the epidermal cells and plays a major role in the tanning process by providing the crucial sclerotizing precursor, NBAD [100]. The ebony gene coding for this enzyme is also responsible for the biosynthesis of carcinine (β-alanylhistamine) by the condensation of β-alanine and histamine in the nervous system mainly in the optic ganglia cells [100]. NBAD synthetase appears to be related to the family of the microbial non-ribosomal peptide synthetases and has three functional domains: activation domain, thiolation domain, and putative amine-selecting domain [100]. The reaction catalyzed by NBAD synthetase requires ATP, β-alanine and dopamine. The coupling reaction resembles very much the non-ribosomal peptide synthesis [100]. First the inactive form of the apoenzyme has to be converted to active holoenzyme form by the addition of 4-phosphopantetheinyl group of coenzyme A on to the conserved S611 present at the thiolation domain of the enzyme by 40-phosphopantetheinyl transferase. The thiol group thus introduced into Int. J. Mol. Sci. 2016, 17, 1753 17 of 24 the holoenzyme plays a pivotal role in the catalysis. At the activation domain, β-alanine is adenylated by ATP and the resultant activated β-alanine is transferred to the thiol group. The terminal group of dopamine bound at the amine-selecting domain then attacks the carbonyl group of β-alanine present at the thiolation site resulting in the biosynthesis of NBAD [100]. Ebony mutant flies have a very soft cuticle with a very dark color [101]. Since NBAD is not synthesized in the mutant flies, their cuticular sclerotization is dramatically affected. The unused dopamine is shunted for melanin production and hence these flies have a dark colored but soft cuticle. Tan is another crucial gene associated with the melanogenic pathway in the cuticle [102,103]. This gene codes for NBAD hydrolase, which is responsible for releasing dopamine back from NBAD. This ensures the sufficient utilization of NBAD for cuticular sclerotization and then allowing the remaining NBAD be converted back to dopamine for melanin biosynthesis. Therefore most likely the melanin made in cuticle is dopamine melanin and not dopa melanin. This is in sharp contrast with the melanin made in the skin of higher animals, which is mainly dopa-derived melanin. In the photoreceptor neurons, NBAD hydrolase has a different role by hydrolyzing carcinine and regenerating histamine necessary for normal vision [102]. NBAD hydrolase closely resembles a family of cysteine peptidases found in fungi. Notably it exhibits high sequence similarity to isopenicillin-N-acyltransferase, which is responsible for the synthesis of penicillin G. This enzyme possesses two activities—a hydrolase and an acylase activity [102]. NBAD hydrolase activity is similar to the first one. In addition, NBAD hydrolase processing is also similar to isopenicillin-N-acyltransferase processing. Thus, the tan gene product is made as an inactive protein, which is self-processed at Gly121-Cys122 to form α,β subunits. Neither of them individually are active but the heterodimeric α,β-pair is biologically active [103]. The black gene encodes for aspartate decarboxylase, which catalyzes the formation of β-alanine from aspartate [104]. β-alanine is required for the formation of the sclerotizing precursor, NBAD by the ebony gene. It should be noted here that β-alanine can also be formed by uracil hydrolysis and that some insects depend on this pathway in supplying β-alanine at the larval stage. However, the majority of β-alanine seems to arise by the action of aspartate decarboxylase [105]. Association of β-alanine to insect pigmentation was known for a long time, but only recently, the molecular biological aspects associated with the process are coming to light. The knockdown of aspartate decarboxylase by double stranded RNA in the beetles Tribolium castenium led to the formation of dark cuticle compared to the rust-red color wild type [105]. Similarly, black pupal mutant in the silkworm, Bombyx mori was shown to be due to frame shift mutation in aspartate decarboxylase gene [106]. Therefore, similar to NBAD synthetase mutant, black mutant flies exhibit the dark black color phenotype due to the accumulation of dopamine that is not used for NBAD synthesis but is used subsequently for dopamine melanin synthesis. Laccase belongs to the group of multicopper oxidases found in a variety of organisms [107,108]. They carry out one electron oxidation of their substrates and use the extracted electrons to reduce molecular oxygen into water. Laccases have four copper ions, which are distributed in two different sites. The type 1 site (T1) carries one copper ion and is responsible for substrate oxidation. The type 2 site (T2/T3) has tricopper cluster and is associated with oxygen reduction [107,109]. Both o-diphenoloxidases and laccases are responsible for melanogenesis in insects. The former is probably responsible for melanization observed in the blood and wounding sites while the latter is involved in pigmentation and sclerotization of cuticle. Recent studies show that laccases are vital for the oxidation of catecholic compounds in insect cuticle [76,108,110–113]. The RNAi experiments carried out in Tribolium castenium attested the key role played by laccase 2 in sclerotization reaction in this beetle [76]. The presence of two spliced isoforms of laccase (laccase 2A and laccase 2B) has been reported in many insects including Manduca sexta [110], Tribolium castaneum [112], Anopheles gambiae [112], and Bombyx mori [113]. Interestingly, the laccase 2 isoforms exhibit no significant differences in their substrate specificity [112]. Futahashi et al. [111] demonstrated that laccase 2 and not tyrosinase 1 or 2 catalyzes the first step in both sclerotization and melanization pathways operating in the cuticle of stinkbugs. Insects injected with dsRNA for tyrosinase 1, tyrosinase 2, and laccase1 had pigmented and sclerotized cuticle and had no lethality whereas the ones injected with dsRNA for Int. J. Mol. Sci. 2016, 17, 1753 18 of 24

Laccase 2 had a soft and colorless cuticle and their adults died within few days after eclosion [111]. The purification and characterization of cuticular laccases has presented considerable challenges to scientists working in the area. Often cuticular laccases are tightly bound and require proteolytic treatment to release [113]. Yatsu and Asano [113] reported that cuticular laccase from Bombyx mori is extractable with urea but requires activation by trypsin. Thus, it is possible that the cuticular laccases are present in the inactive zymogen form and need activation by proteases. However, studies by Dittmer et al. [110] on the recombinant Manduca sexta protein show that the laccase is active with out protease treatment. DCDT is one of the important enzymes of melanogenesis. DCDT has been purified and characterized from many insects since it was first discovered in Manduca sexta [4,21–23,46,57–59]. Importantly, DCDT is also associated with the innate immune response of insects like tyrosinase [4,114–117]. Activity of DCDT increases in response to microbial infections in Drosophila melanogaster [116] and injection of Sephadex beads into mosquitoes [114]. The infection of A. gambiae by malarial parasite causes an increase in the transcripts of DCDT [117]. DCDT belongs to a group of proteins called yellow gene family [118]. In Drosophila melanogaster, fourteen yellow related genes have been recognized. The first yellow gene identified is associated with cuticular melanization and is named yellow-y [118]. The yellow-y gene has been extensively studied but the function of the protein encoded by this gene remains still undetermined. Yellow protein is a secreted protein that has emerged from Royal Jelly protein and seems to be multifunctional [118]. It is expressed in larval salivary gland, in the nervous system of adults, and is associated with the black pigmentation in the adult flies. Mutation in this gene is responsible for the formation of yellow to brown coloration of the cuticle and hence the name yellow [118]. There are many hypotheses regarding the function of the yellow-y protein and how it promotes dark pigmentation in the literature. However many of these suggestions are not supported by experimental verifications. Walter et al. [119] evoked that it is a structural protein that is required for crosslinking DHI. However, to date even in mammalian system, no one has identified such a functional protein. Wittkopp et al. [80,120,121] suggested that yellow protein works downstream of dopa in melanin biosynthesis but the site of action still remains a mystery. Han et al. [46] expressed the yellow-y protein successfully in insect cell line, but were unable to provide support that it catalyzes DCDT reaction. However, their studies successfully identified yellow-f and yellow-f2 as DCDT [46]. Drapeau [122] proposed that yellow-y is a hormone or growth factor like protein that induces melanin formation by activating a signaling pathway. However, again experimental evidence for such a proposal is still missing. Thus, in spite of the enormous work carried out on the yellow gene family, its relation to cuticular melanogenesis remains still unexplained. Further studies are desperately needed to resolve this aspect.

14. Summary and Conclusions A simplistic view of melanogenesis was delineated in the early part of last century by the pioneering work of Raper and Mason. Extensive studies carried out by numerous laboratories resulted in the discovery of several proteins and enzymes associated with the melanogenic pathway. Many research groups elucidated the function of many genes encoding the regulatory proteins and/or enzymes involved in the pigmentation pathway. Intensive investigations on the biochemistry and mechanism of transformation of various catecholamine derivatives resulted in the identification of novel reactive transient intermediates. From these studies, a clear and detailed picture has emerged not only on the biochemical aspects but also on the molecular biological aspect of melanogenesis. We are beginning to see some unity and variations in the scheme of reactions used by this process in different groups of organisms. Some important uncertainties are still unresolved. How is DHI biosynthesized in higher animals? Do insects and other arthropods make significant amounts of pheomelanin and DHICA melanin? Why are two different types of dopachrome converting enzymes present in the two major groups of animals? How are various enzymes regulated to produce different shades of color? Are there any new enzymes/proteins to be discovered? Since melanin production in animals is Int. J. Mol. Sci. 2016, 17, 1753 19 of 24 associated with a number of pathological conditions, more research is certainly needed. Unraveling the role of many genes associated both with both the melanogenic process and sclerotinogenic process could be extremely helpful to treat some of the associated diseases. Finally, a note on the hormonal regulation of melanogenesis in mammalian and insect systems warrants some attention. We are beginning to understand some aspects of hormonal regulation of melanogenesis in mammalian system [123–125]. Even melanogenic precursors, tyrosine and dopa seem to act as hormone-like regulators of melanogenic pathway in mammals [126]. However, in insects, the hormonal regulation of both melanogenic and sclerotinogenic processes need to be explored in details to shed more light on the physiological, biochemical and genetic aspects of these two processes. With the advent of molecular biology tools, we are certain that some of these aspects will be brought to light in the near future.

Acknowledgments: We thank Geetha Sugumaran of Veterans Administration Hospital, Boston, MA 02130, USA for her critical evaluation of the manuscript and useful suggestions. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

NADA N-acetyldopamine Dehydro NADA 1,2-Dehydro-N-acetyldopamine DHI 5,6-Dihydroxyindole DHICA 5,6-Dihydroxyindole-2-carboxylic acid NBAD N-β-alanyldopamine Dehydro NBAD 1,2-Dehydro-N-β-alanyldopamine DCT Dopachrome tautomerase DCDT Dopachrome decarboxylase/tautomerase

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