Microbial Tyrosinase: Biochemical, Molecular Properties and Pharmaceutical Applications

1Enzymology and Fungal Biotechnology lab, Botany and Microbiology Department, Faculty of Science, Zagazig University, Zagazig, 44519, Egypt. 2Chemistry Department, Faculty of Science, Zagazig University, Zagazig, 44519, Egypt. 3Botany and Microbiology Department, Faculty of Science, Benha University, Benha, 13518, Egypt. 4Faculty of Biotechnology, October University for Modern Science and Arts, Cairo 12566, Egypt.


Biological identity and types of tyrosinases
Tyrosinase (EC 1.14.18.1) is a coppercontaining monooxygenase involved in production of O-quinones (diphenolase) through catalyzing the o-hydroxylation reaction of monophenols to produce O-diphenols (monophenolase) which upon oxidation gives O-quinones as reported by Sanchez-Ferrer et al., (1995) (Fig.  1). Tyrosinase is mainly involved in the synthesis of melanin via two reactions. Firstly, upon O-hydroxylation reaction tyrosine transferred into 3,4-dihydroxyphenylalanine (L-DOPA) which undergoes further oxidation to produce dopaquinone, that finally by many enzymatic processes produces melanin. Practically, in microorganisms the melanin's biosynthetic pathways are a little bit different from that of human cells. Funa et al., (1999) reported that in fungi, melanin can be synthesized from 1,8-Dihydroxynaphthalene (DHN) precursors (DHNmelanin following polyketide pathway, using malonyl-CoA as a helper factor (Alspaugh et al., 1998). In human and few microbes, melanin pigments were synthesized by L-DOPA pathway using the precursors molecules of tyrosine that hydroxylated into L-DOPA then oxidized into dopaquinone and into further melanin pigment (Butler and Day, 1998). The melanin biosynthetic scheme in fungi has been illustrated in Fig.  1. Melanin is amorphous polymer, negatively charged and results from many enzymes catalyzed processes and autoxidative polycondensation of various hydrophobic groups in quinone (Jacobson, 2000, Langfelder et al., 2003, Ito and Fujita, 1985. Melanin pigments has the ability of deactivating the free radicals, peroxides, absorbing heavy metals, thus exhibits profound antioxidant activity (Olennikov et al., 2012;Ró¿anowska et al., 1999;Cunha et al., 2010). Melanin is characterized by the presence of mobile ð-electrons giving it extraordinary electronic properties and enabling it as a powerful protector against UV-and solar radiation (d' Ischia et al., 2009). Pigments of melanin can absorb light in a wide range, their absorption intensity ability increases gradually with decreasing wavelengths (Meredith and Sarna, 2006). Depending on the heat results from photon energy's conversion, melanin can absorb light (Riesz et al., 2006), which may result in protons transfer inside a monomer because during the photoexcitation of the pigment energy can be released (Olsen et al., 2007). Melanin's photoionization and its subsequent partial destruction can be noticed after the interaction with the UV radiation in the range of (240-300 nm). Thus, during UV radiation cytotoxic byproducts can be formed despite the effective protecting roles of cellular melanin against both UV-and solar radiation. In melanin synthesis, a multi-copper oxygenase enzyme called tyrosinase is a rate limiting enzyme contains binuclear active sites for Copper (Garcia-Jimenez et al., 2017) Tyrosinases have been well-characterized from many microorganisms as Streptomyces glausescens, Neurospora crassa and Agaricus bisporus (Kupper et al., 1989). It was reported that the melanin deficiency of oculocutaneous albinism was the main reason for human tyrosinase's mutations (Oetting and King, 1994). In human, the possible role of melanins is to prevent the pathogens to enter the wound which may results in healing of wound and the cuticle possibly can be sclerotized. While, in fungi the main role of tyrosinase is catalyzing the first step in the melanin pigment formation from tyrosine. The bacterial tyrosinases involved in melanin production was reported to be extracellular enzymes (Claus and Decker, 2006)

Melanogenesis
Melanins which produced by melanocytes (a membrane-bound granule), are the main pigment responsible for pigmentation of human's skin, hair and eyes (Kim and Uyama, 2005) through melanogenesis. The number of melanosomes increases with continuous exposure to sunlight resulting in an increase their melanin content and final it transfers to keratinocytes. Skin pigmentation and melanogenesis are the main photo-protective factor responding to the bad effect of UV radiation from the skin and sun photo-carcinogenesis and removing reactive oxygen species (Mohania and Chandel et al., 2017). Melanogenesis process is a very complicated pathway consists of a mix of both chemically and reactions catalyzed by enzymes. Two different types of melanin can be produced by melanocytes through linkage with cysteine or glutathione: pheomelanin (red-yellow) and eumelanin (brown-black) (Fig. 3) Pillaiyar et al., 2015). The process of melanogenesis is started by L-tyrosine oxidation to prduce dopaquinone by tyrosinase enzyme and the eumelanin and pheomelanin will be synthesized from the resulted quinone which used as a precursor for the process of synthesis. In melanin synthesis, the formation of dopaquinone is a rate-limiting step of because at a physiological pH value the rest of steps can occurs spontaneously (Halaban et al., 2002). Indoline, leukodopachrome (cyclodopa) can be produced after formation of dopaquinone, because during this intramolecular cyclization must be occured. Both of dopachrome and L-3, 4-dihydroxyphenylalanine (L-DOPA) can be increased because of redox exchange between leukodopachrome and dopaquinone respectively, which is another substrate for TYR and gives dopaquinone through oxidation by tyrosinase. TRP-2 (dopachrome tautomerase (DCT)) catalyzes the decomposition of Dopachrome to produce dihydroxyindole (DHI) and dihydroxyindole-2carboxylicacid (DHICA). Finally, eumelanin can be produced through the oxidation of the produced dihydroxyindoles (DHI and DHICA) by the action of TRP-1. In the same line, 5-S-cysteinyldopa or glutothionyldopa are produced from dopaquinone conversion in the presence of cysteine or glutathione. Further oxidation results in production of pheomelanin at end. Tyrosinase is very necessary for melanogenesis despite the involvement of the three different enzymes, TYR, TRP-1 and TRP-2 in the melanogenesis pathway.
Melanocyte is the only cells that can produce tyrosinase in the Golgi and endoplasmic reticulum, following its production it transferred to melanosomes, for synthesis of the pigment melanin. Tryosinase enzyme contains three histidine residues surrounding two ions of copper giving tyrosinase its catalytic activity of. During the formation of pigments the active sites are characterized by three states named; oxy, met and deoxy forms (Fig. 4). The specificity of tyrosinase enzyme backs to the fact that at the enzyme active site, two copper ions interacts with dioxygen to produce an intermediate which is highly reactive participating in the hydroxylation reactions of monophenols to diphenols (monophenolase activity) and in o-diphenols oxidation to o-quinones (diphenolase activity) (Decker and Tuczek, 2000).
Many studies has widely focused on monophenolase mechanism for tyrosinase activity substrates which increases diphenolase activity (Wilcox et al., 1985). It is conceivable that for o-hydroxylation the monophenolic substrates require a rearrangement from the axial to equatorial arrangement for simple electron transfer, while, the o-diphenolic substrates doesn't need like this rearrangement at the copper site. Catalytic activity of tyrosinase on monophenols is lower than on o-diphenols as the kinetic studies of the steady state showed (Cabanes et al., 1987). The activity of monophenolase is characterized by a lag time (Fenoll et al., 2001) in which the concentration of both substrate and enzyme with the presence of hydrogen donor are a limiting factors (Cooksey et al., 1997). In the kinetic studies, lag time is the time needed for the resting met form to be transferred into the active deoxy form in presence of a reducing agent, results through the action of the little amounts of the oxy form that found with the met form. In the presence of cofactors which is a reducing agents, especially the derivatives of o-diphenol such as L-DOPA and (+)-catechin, tyrosinase can be activated and the lag time was shortened or totally abolished (Fig.5). At a very low concentration, L-DOPA is the reducing agent which effectively can eliminates lag time. The process of neuromelanin production can be catalyzed by tyrosinase which catalyzes the oxidation of dopamine producing dopaquinones. This findings suggests that a significant role might be play by tyrosinase in the formation of neuromelanin in the human brain cells and responsible for the neurodegeneration associated with Huntington's diseases and Parkinson's disease . Thus, melanin synthesis regulation through tyrosinase inhibition can be a good motivation for researchers concerned with hyperpigmentation prevention.

Crystal structure of Tyrosinases
The structural properties of tyrosinase are different in nature with the distribution in different sources (Jaenicke and Decker, 2003;Mayer, 2006). The structural variations of tyrosinases are mainly in amino acid sequences, molecular configuration, size, posttranslational modification mechanisms, and active site structure. Tyrosinases are composed of two ions of copper (Cu A and Cu B) each attached by six histidine residues in their active site. The crystal structure of a tyrosinase was identified for the first time in plant in its active form was purified from the leaves of walnut and showed activity for both monophenolase and a bulky residue at the blocker position (Zekiri and Molitor et al., 2014). Walnut tyrosinase (jrTYR) was purified from leaves of walnut and the X-ray crystallography was used to determine structure to a 1.8 Å resolution, showing identical structure with polyphenol oxidase (PPO) Mauracher et al. 2014). It shares a higher similarity in the structure with Ipomoea batatas catechol oxidases (Klabunde and Eicken, 1998) and Vitis vinifera (Virador et al., 2010). The region of active-site which contains in its center a binuclear copper is formed of a bundle of four helices (á4, á5, á12, and á14). Each copper ion in the active-site is fixed by three histidine residues (His) (Fig. 3). Copper A (CuA) is coordinated by His87, His 108 , and His 117 , where His 87 and His 117 are located on á-helices (á4 and á5) and His 108 on a loop. A thioether bond is formed from the C atom of His 108 in with the sulfur atom of accompanied cysteine (Cys 91 ), giving a limited flexibility of His 108 . Copper B (CuB) is arranged by His 239 , His 243 , and His273, located on á-helices. A hydroxide anion serves as A Bridge between the two copper centers which are 2.1 Å from each copper ion. The N terminal loops can be stabilized by two disulfide bonds (Cys 11 -Cys 26 and Cys 25 -Cys 88 ), anchoring them to the main core, and copper incorporation (Fujieda et al., 2013). In jrTYR the two disulfide bonds are located at a distance of about 8 Å next to each other. A phenylalanine residue (Phe 260 ) in jrTYR is located at the position of blocker residue above CuA. It has been suggested that in catechol oxidases the bulky phenylalanine prevents the substrates from binding to CuA, leading to the lack of monophenolase activity (Olivares et al., 2002;. The absence in activity of monophenolase in catechol was suggested to be due to the limited flexibility of the CuA site in combination with a bulky residue of oxidases, since these restrictions will prevent the rotation of substrate, which is necessary for the hydroxylation of monophenols (Goldfeder et al., 2014).

Molecular, Biochemical properties and sources of Tyrosinase
The molecular subunit structure of tyrosinase was slightly varied with the biological identity of the producing microbes. Tyrosinase had a homodimer subunit structure of 50 kDa from Aspergillus nidulans (Birse and Clutterbuck, 1990), Agaricus bisporus (Lopez-Tejedor and Palomo, 2018) and Trichoderma reesei (Selinheimo et al., 2007). The structure of molecular subunit of tyrosinase from Streptomyces spp was 30kDa (Wu et al., 2010). The enzyme Mycothermus thermophiles had an approximate native molecular mass 320 kDa, with subunit structure 80 kDa on the SDS-PAGE, revealing their composition of four identical subunits (Sutay Kocabas et al., 2008). Also, the enzyme from Thermomicrobium roseum has two identical subunits with 43 kDa .  (Liu et al., 2004). The pH 9.5 was the optimum for tyrosinase of Thermomicrobium roseum, the enzyme loss about 75% its of activity at pH 6 but retains full activity upon standing 20 h in the pH-range 8.5-10.0 . The purified enzyme optimal pH of T. reesei was 9 (Selinheimo et al., 2006). The highest activity of tyrosinase from A. bisporus was at pH 7.0 (Zaidi et al. 2014), while, the enzyme optimal pH from Streptomyces sp was 6.8 (Yoshimoto et al., 1985). The maximal activity of tyrosinase from Streptomyces pharetrae and Streptomyces polyantibioticus was reported at  Table 1.

Biotechnological applications of tyrosinases Pharmaceutical applications
Tyrosinase can be found in macro organisms as animals, plants and microorganisms . Tyrosinase in human's cell, is a very important step which regulates the melanin production in the melanosomes of melanocyte cells resulting in pigmentation of skin, hair and eyes giving the protection against Ultra Violet light (Videira et al., 2013). In addition, tyrosinase playing a pivotal role in immune response and healing of wounds (Marino et al., 2011). Tyrosinase has an important medical application including L-DOPA production, a drug used in the treatment of the Parkinson's disease, and other neurological diseases (Valipourand and Burhan, 2016). Tyrosinase is used for production of hydroxytyrosol as an estrogenic intermediate (Zhang et al., 2007), treatment of vitiligo (Seo et al., 2003).

Food Industry
Tyrosinase has been implemented in various food processing by production of various food additives (Valipour and Burhan, 2016). Tyrosinase has been frequently used as emulsifiers in manufacturing natural polymers can be cross linked for the production of new polymers (Faria et al., 2007), based on the accessibility and abundance of target protein (Heck et al., 2012). Tyrosinase can be used to yield a novel bioproducts through grafting of specific compounds to biopolymers. The byproducts of food processes can be converted to environmentally valuable items by tyrosinase (Aberg et al., 2004). Tyrosinase as cross-linking enzymes are exploited in tailoring the gelation properties of foods, that has been characterized as a highly specific agent in the reaction they work on, in addition to utilizing food matrix components like proteins (Selinheimo, 2008).

Enzymatic browning of plant-derived foods
The fruit and vegetables browning have gained a high attention in the field of food processing, as it lower its economic price. Browning by enzymes is a main factor in fruits damaging during the process of post-harvest and handling, because of mixing tyrosinase enzyme and their polyphenolic substrates after crushing operations leading to rupture of cell structure (Yi et al., 2010). Tyrosinase oxidize the phenolic compounds in fruits leading to bad changes in flavor, color and texture, thereby reducing its price in markt (Xu and Zhang et al., 2017). The browning process depends upon many factors, such as enzyme concentration and substrate, availability of oxygen, temperature and pH (Zheng et al. 2008).
The process of enzymatic browning was reported to start with O-diphenol such as L-DOPA transformation to O-quinone, which produces brown melanin pigment, upon further oxidation (Busch, 1999). Several chemical and/or physical agents have been used to control the browning by enzymes such as blanching, chemicals application, high temperature and microwave (Ioannou andGhoul, 2013, Zhang et al., 2018b). Unfortunately, these processes are characterized by many disadvantages as changing of nutritional quality and organoleptic of the end products, representing a potential risk for human health (Tinello and Lante, 2018). Therefore, several enzyme inhibitors, namely ascorbic acid (Kubglomsong et al., 2018), for browning prevention citric acid and kojic acid, have been used (Friedman, 1996).

Tyrosinase inhibitors
Tyrosinase can be overexpressed by the inner factors as hormones which stimulate melanocyte-and other environmental factors such as irradiation by UV, which are the main reason of  et al., 2016). Therefore, tyrosinase inhibitors attract the attention of various researchers, especially for medical and cosmetic applications. Several inhibitors for tyrosinase as kojic acid and hydroquinone may be effective in clinical applications and formulations in topical use (Fujimoto et al., 1999). Generally, in the presence of a monophenolic or a diphenolic substrates these inhibitors are examined and the enzyme activity was measured based on dopachrome produced (Lin et al., 2008). The inhibitors mainly contain copper-binding agents and competes for binding with the active sites on tyrosinase (Robb, 1984). Analogues of the main substrate include numerous aromatic acids, phenols and their derivatives can be used as a competitive inhibitor (Nicolas et al., 1994) to tyrosinase. Because the metalloproteinic identity of tyrosinase, metal chelators such as thiourea derivatives, kojic acid, carbon monoxide, cyanide, tropolone, could be used as enzyme inhibitor. Several natural compounds with anti-tyrosinase activity have been recently recommended because of their lower

Plants secondary metabolites
Flavonoids are polyphenol derivatives containing pyrane rings and phenols frequently found in the seeds, leaves, bark and plant's flowers. Over the identified 4000 flavonoids, these compounds mainly participate in the protection of plant against its pathogens and UV radiation (Harborne and Williams, 2000). Flavonoids are the largest groups of natural inhibitors of tyrosinase (Wang et al., 2014) that are classified into six groups: flavanols, flavones, flavonols, flavanones, isoflavones and anthocyanidins. Most of flavonoids are competitively inhibit tyrosinase by binding with tyrosine. Some of flavonoids, such as catechin, rhamnetin, kaempferol, quercetin and morin are analogue of tyrosine that competitively inhibits tyrosinase activity (Kim 2013, Kubo et al., 2000, Xie et al., 2003. Steppogenin, is a flavanone derivative, isolated from the Cudrania tricuspidata twigs, showed a potential inhibition to tyrosinase activity by about 10-times greater than kojic acid (Zehng et al., 2013).
Aloesin, a hydroxychromone glucoside purified from Aloe vera. It has been shown to be a potent and selective tyrosinase's inhibitor which has direct inhibitory effects on melanogenesis (Jin and Lee, 1999). After UV radiation aloesin may be able to inhibit the hyperpigmentation in a dosedependent manner (Choi et al., 2002).
Coumarins are heterocyclic compounds naturally occurs, consists of an aromatic ring connected to a condensed six-member lactone ring. Both coumarins and their derivatives are characterized to be biologically and pharmacologically active especially as inhibitors for tyrosinase (Bubols et al., 2013;Wu et al., 2012 andHassan et al. 2018). Esuletin, dihydroxylcoumarin isolated from Euphorbia lathyris, displaying a potential tyrosinase inhibition. Other coumarin analogs, 9-hydroxy-4-methoxypsoralen, isolated from Angelica dahurica (Ahmad et al., 2004). From the molecular Gallic acid (3,4,5-trihydroxybenzoic acid) is a polyphenol compound displaying a potential inhibitory effect to tyrosinase activity (Kubo et al., 2000, Lu andNie et al., 2006) by reducing dopaquinone back to L-DOPA through a redox reaction. Gallic acid and its dervitives esters are used in the field of food industry as additives. Gallic acid prevents the oxidation reaction of L-DOPA enhanced by tyrosinase (Masuda et al., 2008). Most of gallic acid derivatives have been isolated from green tea and Galla rhois (Khan et al., 2006).
A n t h r a q u i n o n e s h a v e v a r i o u s pharmacological applications especially antityrosinase activity that being more powerful than kojic acid (Leu et al., 2008). Among the natural anthraquinones, benzaldehyde, benzoic acid, anisaldehyde, cinnamic acid, anisic acid and vanillic acid (Maghsoudi et al., 2013, Nihei andKubo, 2017). These compounds displaying noticeable inhibitory activities towards tyrosinase. For example, 3,4-dihydroxybenzaldehyde-Oethyloxime (IC 50 0.3 µM) had a potent tyrosinase inhibitory effect that being consistent to the authentic inhibitor, tropolone (IC 50 0.13 µM) (Ley and Bertram, 2001). The aldehyde group and terminal methoxy group in derivatives of benzaldehyde playing an important role on its effect as inhibitor by chelating the copper ion present in tyrosinase's active site (Conrad et al., 1994).

Fungal secondary metabolites
Fungi produce a plethora of bioactive compounds especially with potential inhibitory effect to tyrosinase (Criton et al., 2008).
Among these metabolites, kojic acid has been reported as the most powerful "wellknown" inhibitors to tyrosinase activity. Kojic acid (5-hydroxy-2-hydroxymethyl-ã-pyrone) has been used commonly as skin-whitening cosmetic (Da Costa et al., 2018) and as an additive in food for inhibition of browning by enzymes (Bentley, 2006). Kojic acid works as as a bidentate chelator for the reaction of metal ions transition such as Cu +2 and Fe +3 , with an inhibitory effect to the monophenolase and diphenolase enzyme activity of tyrosinase frommushroom (Kim and Uyama, 2005). Nevertheless, the usage of kojic acid in cosmetics is limited because under the light it's unstable and also, in aerobic condition, thereby; many trails for creating synthetic kojic acid derivatives were reported aiming to improving the ability metal chelating and structural stability (Saghaie et al., 2013). Kojic acid was frequently produced by A. albus, A. candidus, A. niger and Penicillium sp (Wei et al. 1991).
Azelaic acid (1, 7-heptanedicarboxylic acid), which is a saturated dicarboxylic acid in a straight-chain, that mainly obtained from yeast, Pityrosporum ovale through lipoperoxidation of free and esterified cis-polyunsaturated fatty acids. This acid is a cytotoxic agent on malignant melanocytes of primary cutaneous melanoma, while has no effect on normal melanocytes (Schallreuter and Wood, 1990).
Yeast metallothioneins are ubiquitous cytosolic proteins with a selective binding to heavy metal ions (Zn 2+ , Cu 2+ and Cd 2+ ) and cysteine (Hamer, 1986). It was reported that Neurospora crassa copper-metallothionein is a metal donor for apo-tyrosinase. Metallothionein from Aspergillus niger was reported as an inhibitor for various mushroom tyrosinases.

Chemically synthesized tyrosinase inhibitors
Captopril ((2S)-N-(3-mercapto-2methylpropionyl)-L-proline) is a drug has a wide application in the hypertension treatment and heart failure due to its effect in inhibition of the angiotensin-converting enzyme (Cleland, 1994). This drug is characterized by irreversibly inhibition to monophenolase of tyrosinase from mushroom, although, it exhibits irreversible competitive inhibition to diphenolase of tyrosinase (Kuo and Ho, 2013). Inhibition of monophenolase and diphenolase activities of tyrosinase by captopril showing a positive kinetic cooperativity. Also, Captopril is known as a copper chelator, so, its inhibitory mechanism could be rationalized by chelating copper ions at the active site of tyrosinase. As well as, Tropolone (2-hydroxy-2,4,6-cycloheptatriene) is one of the most potent tyrosinase inhibitors (Ismaya and Rozeboom, 2011). It is analogous in its structure to tyrosinase's o-diphenolic substrates as well as an effective copper chelator.
Resorcinols such as m-coumaric acid, L-mimosine and 4-substituted resorcinols has also been reported with slow-binding inhibition of tyrosinases from different sources (Garcia-Jimenez et al., 2016). However, resorcinol is a poor tyrosinase inhibitor, substitution in the 4-position yields increased inhibitory activity. The highest inhibition was obtained with hydrophobic substituents in the 4-position such as 4-hexyland 4-dodecylresorcinol (McEvily et al., 1992). Hexylresorcinol was reported as an effective inhibitor to be used in the food industry due to its characters which includes, water solubility, stability, nontoxicity, non-mutagenicity and non-carcinogenicity, and it has been used in the prevention of shrimp melanosis because it recognized as safe and also, in controlling of browning in fresh and hot-air-dried apple pecies as well as potatoes and avocados.
Hydrogen peroxide is reported as inhibitors of diverse enzymes which contain copper-such as dopamine b-monooxygenase and tyrosinase produced by mushroom (Andrawis and Kahn, 1985;Connor et al., 2011). Activities of both monophenolase and diphenolase of mushroom tyrosinase can be lost on exposure to high concentration of H 2 O 2 . In the presence of relatively high concentrations of H 2 O 2 , tyrosinase is converted to an oxy-oxytyrosinase (inactivated form of tyrosinase), which is a hypothetical intermediate analogous to the ternary complex formed between oxytyrosinase and o-dihydroxyphenol.
In conclusion, the biochemical, molecular and structural properties of tyrosinases from different microbial sources has been extensively documented. The potential chemical inhibitors of different biological identities for tyrosinases have been addressed that could be used as anti-melanin hyperpigmentation as well as in treatment of neuromelanin disorders.