Identification of a tyrosinasse from a periphytic marine bacterium

275

inase from a periphytic

~cterium

• C o y n e *, D a r r e n D. Sledjeski, W. q a n d R o n a l d M. W e i n e r

aqua

~obiolog), Department, Universityof Maryland, College Park. and Center ofrM, Marine BioteehJ

MD, U.S.A.

Received8 September 1989 Accepted 17 October 1989 Key words: Tyrosinase; Melanin; Bacterial Bacteri pigmenl

4MARY ewly isolated periphytic marine bacterium en shown to synthesize a true tyrosinase, zyme exhibited both cresolase and catechonctions and catalyzed the biosynthesis of n from t.-tyrosine. Enzyme activity was enhanced in the presence of oxidants and was inhibited by copper chelating agents such as diethyldithiocarbamic arbamic acid and cyanide. The apparent molecular lar weight of the 2-40 tyrosinase (67000) makes this enzyme the largest known procaryote tyrosinase.

2. I N T R O D U C T I O N

[6], Pseudomonas PseudoJ [7], S [8], Bacillus [9], Legionella Legione [10] an, 21. True tyr crosinases ( ) are internal monooxygel mases that zymatic activities, namel$y a cresola ~holase, which are intimate intimately coupled to mediate the conversion con of tyrosine via L-DOPA to dopachrome ~chrome. Once dopachromc 3achrome is formed, the other reactions eactions in the bacterial melanin m biosynthetic pathway are a~ very rapid and probably p nonenzymatic [2,13,14] 2,13,14]. The marine mar bacterial isolate 2-40 [15] sythesizes a black blac pigment at the late exponenl ~onential-stationary phase pha of growth. The polarly-fla! arly-flagellated bacterium i,is Gram-negative, rod-shaped, and al possesses a G C + C content of 45.66 mol~. moll The bacterium,,which is able to degrade a number nun of polysaccharides, is most closely related to t¢ members of the genus Alteromonas [15]. This paper de~ribes the nature of the tyrosinase acti, activity associated with the marine bacterium 2-40 2-~ and identifies the black pigment as a melanin. •

Melanin pigments occur widelly in plants and animals, and often in bacteria [1]. 1]. While maremalian melanogenesis has been extensivel tensively studied [2], much less is known concernin ng bacterial pigment formation. Bacterial genera in in which melanin production has been described include elude Aeromonas [3], Azotobacter [4], Mycobacterium ium [5], Proteus

.

MATERIALS A N D METHODS 3.1. Organism and growth conditions

fiology Department, Correspondence to: V.E. Coyne, Microbiolo~ Universityof Maryland,College Park, MD D 20742. 20742, U.S.A.

A bacterium, designated strain 2-40, was obtained from G. Andrykovitch, George Mast Mason Uni-

0378-1097/90/$03.50 © 1990 Federation of European MicrobiologicalSocieties

salt marsh s growing salinity, ; in Difco final conhe carbon icroorgan-

i 2-40 was grown as described for 68 h. ,'-log phase culture (500 ml) was washed 0.1 M Na2PO4 buffer, pH 6.4, and subseresuspended in a final volume of 10 ml Cell lysates were prepared by sonication onifer cell disruptor 350 at power setting 7 duty cycle for 4 rain. The cell lysates d stable at - 7 0 ~ . Tyrosinase enzyme acis estimated by a modification of the assay ed by Pomerantz and Murthy [16] whereby formation was determined visually after at 42°C. The assay mixture typically conFthe following: L-tyrosine, 4 mM (saturatt respect to the enzyme); 2-40 cell lysate, and 10 mM phosphate buffer, pH 6.4 in a lume of 1 ml. Mushroom tyrosinase (Sigma Chemical ~1 Co., St. Louis, MO) provided a positive control against which the level of melanin produced b~y the 2-40 tyrosinase enzyme was estimated.

3.3. Physical conditions The optimum physical conditions for 2-40 tyrosinase Lse activity were determined by measuring the rate of melanogenesis between pH values of 6 to 8 at 25°C and the effect of tern :mperatures between 25°C and 95°C at constant nt pH. The influence of the osmolarity of the :he buffer was determined at constant pH and tem[ mperature. 3.4. Substrates The specificity of the tyrosinase se enzyme from strain 2-40 was determined by measunn ~asuring melanin production from D-tyrosine, L-DOPA• OPA, L-phenylalanine, m-cresol, p-cresol and catechol. Each substrate was tested at a final concentrati ncentration ration of 4 mM which was saturating with respect to the enzyme.

3.5. Enzyme stimulation The effect of both c melanogenesis from L with ammonium persull gen peroxide (1-880 m

reductants on s determined ~l) and hydroely.

3.6. Enzyme inhibition Inhibition of 2-40 was attempted by trea with either diethyldit (0.1-1 raM), mM), sodium ~toethanol (6.4 Iv mercaptoeth~ mrchased from were purcha: Louis, MO). DDC and were either added 30 neously to, the tl additiox

zyme activity e cell extract acid (DDC) i raM) or 2dl compounds dcal Co., St. dde inhibitors • or simulta-

3. 7. Pigment characteri2 The iden!tity of the from tyrosine tyrosim by the 2ploying various ! by employinl characterization [3].

em produced as determined d for melanin

3.8. Serology Mushroov lMushroom v l u s n r o o m tyrosina.~ t y r o s l l l a s ~ [ ~ i g m a • . n t :~mical m l c a l Co.• • St. Louis, MO) was dissolved in PBS to a final fin~ concentration of ol 2 mg/ml. This was emulsifi¢ dsified 1 : 1 with adjuval iuvant (MPL-TDM emulsion. Ribi Ril IramunoChem iResearch• Inc., Hamilton, MT) and 1 ml injected ssubcutaneously into New Zealand Zealar rabbits twice a week for 3.5 weeks. Serum was w~ collected by heart h~ bleeds five days after th~ the final injection. A 48-h slant sh of strain 2-40 was resuspen rended in 1.5 ml marine marin broth. The cells were pellet, ~elleted and resuspended in 100 /tl of lysing buffer (.~ sodium dodecyl sulfate, 2%; 2-mercaptoethanol ~ethanol, 5%; glycerol, 5~; Tris, 62.5 raM, pH 6.8). SDS-PAGE SDS(10% acrylamide) was performed according g to [17], with 50 /tl sample loaded per well. Gel Gels were electroblotted by the method of Towbin et al. 1181 onto MSI nitrocellulose (Fisher Scientific, Sci~ Washington, DC.). A standard protocol [1 [18] was followed to immunostain the western blots blot~ using anti-mushroom tyrosinase antibodies and an peroxidase conjugated goat anti-rabbit IgG antiserum an! (Boehringer Mannheim Biochemicals, lndianapolndi lis, IN).

atic activity to abolish :ylation to tyrosinase ~ound from of melanin r,.40). Since xidized to Irome ana eventually melanm, it ,t was deo omit this compound from the tyrosinase o as to prevent spurious results, imum tyrosinase activity occurred at pH 6.4 42"C. The pH range for activity was from I to 7.8 and the enzyme was active from up to 70"C. Enzyme activity was not afby the osmolarity of the phosphate buffer .0 × 10 -4 M to 1.0 M. bstrates ~sinases catalyze two types of reactions: se activity whereby the enzyme mediates tho hydroxylation of tyrosine and other ,henols to form o-diphenols, and a subsecatecholase activity which results in the oxygenation ,ation of the o-diphenols to produce oquinones les which are unstable and typically polymerize into melanin [19]. The 2-40 cell lysate was examined ked for each of these sequential enzymatic activities. es. The putative tyrosinase enzyme from marine baceterium strain 2-40 catalyzed melanin sytbesiss from L-tyrosine, L-DOPA, D-tyrosine, pcresol and catechol. The observation that p-cresol, and not ~t m-cresol, acted as a suitable substrate for •me.nogig m ay h ~ e.Ynlaine.d v the. n~iticm melanogenesls may be explained hby the nposmon of the hydroxyl group on the phenol nol ring. Accordingly, m-tyrosine was reported toa be a poor substrate for the V. tyrosinaticus tyrosmase ,rosinase enzyme which oxidized it at only 1.5~ of the rate for L-tyrosine [16]. Unlike the tyrosinase from V. tyrosinaticus, which does not oxidize catechol :hol I161, tyr24o exhibited catecholase activity which enhanced melanin production. Tyrosinase occurs in three different states; namely, mettyrosmase, 3sinase, oxytyro)xytyrosinase is sinase and deoxytyrosinase. Ox, ,-diate during the thought to be a catalytic intermediate

oxidation of both m, substrates [20]. Forma the enzyme from me rate-limiting step in t lase activity. Since 95 has been shown to e~ presence of excess di possible that the incre; sis was a result of the sinase form of tyr,.4o d

ld o-diphenol active form of ; possibly the se or catecho~m tyrosinase 'osinase in the npounds, it is lelanin syntheff the oxytyro:atechol.

4.3. lnductit Inductive effects persuifate and hydrogen pc, ~eroxide on t tY~4o Since tyrrrosinases tion reactions, melanogene ,~nesis was te rong oxidative conditions. Ammon 'ate (5 mM) enhanced melanin I wo-fold. Ammonium persulfate pe~ pr( zes the reaction by increasing increasir the avai )lecular oxygen for the formation forn of c [20,211. Mettyros Tosinase is ir :d to form deoxytyrosina ytyrosinase by em ormed o-dihydroxyphenc yphenols or by e ~lded reductant [22]. Deoxyytyrosinase interacts with molecular oxygen c to prouuc¢ oxytyrosma,, yrosinase. Low concentrati~ concentratmns of hydrogen peroxidee shortened shorte the lag period of tyrosine hydroxylation and stimuiated o-dih)ydroxyphenolase activity of the avocado and mushroom mushr~ tyrosinase enzymes, while whi relatively high concentrations of hydrogen p)eroxide inactivated these tyrosinases [22]. Howeve However. 1-20 mM hydroggen peroxide did not affect tyr24 240 activity, while hhigher concentrations (88 mM-0.88 mM-( M) completely inactivated the enzyme. Hydrogen Hydro[ peroxide is known km to inactivate mushroom ty~ rrosinase hby v the. nrl the fformation of tyrosinase Cu(ll)-O [)-OH produced by an interaction between hydrog hydrogen peroxide and the cuprous ions present in deoxytyrosinase [23]. Tyrosinase Cu(II)-OI5 I-OH could oxidize histidine at the active site of the enzyme resulting in release of Cu 2+ from the active act site and concomitant irreversible inactivation of the enzyme. 4.4 Inhibition of tyr24o activity by DDC, cyanide and 2-mercaptoethanol Tyrosinase enzymes isolated from a variety va of both procaryotic and eucaryotic sources have t an

20]. Thus, hyldithiotyrosinase However, resence of tyr24o acae activity sine were S indicate ~mpetitive osine. 0 pretreated with either DDC or cyanide, e reactivated by the introduction of adCu 2+. Not only did the exogenous copper activity, but resulted in an increase in above that obtained with the untreated In accordance with this observation, it ~ntly reported that although synthesis of enzyme is not dependent on copper, the of active tyrosinase molecules in Neuro'assa is significantly increased when the medium is supplemented with copper [24]. sible that inactivated tyr24o was reactivated icing the DDC- or cyanide-bound Cu 2+ achelated copper ion at the active sites of tme. Additionally, exogenous copper may have activated tivated apoenzymes present in the 2-40 cell extract act, resulting in an increase in the amount of active ttyrosinase molecules. Beta-mercaptoethanol irreversibl ~ersibly inactivated tyr24o, consistant with its effect:t on Neurospora, mushroom, arthropod [20], and Xenopus [25] tyrosinases.

Table l Characterizationof strain 2Parameter Color Solubifityin water Solubifity in 0.1 N NaOH Appearancein water Precipitation with 5 mM FeC Precipitationfrom NaOH by Absorptionspectrum(350-6.~

:rty uble )le eulate pitated pirated haracteristic lks

Precipitation ipitationby Blackberg-W technique

pitated

38000 and 27000 : (Fi~ tyrosinase a~ antibodies , peptide from strain 2-, of 67000, while wh two mi 52 kDa wen were also obs

nti-mushroom with a polylecular weight >f 60 kDa and the molecular

a00E

i

!

1

92.5~ ! Sg~ ..... 691

4.5. Pigment characterization The characteristics of the pigment synthesized t w r ~ e ; n ~ by I ~ tyr2~ t w r 0 are ~ r ~ summarized e , , m m alarized r~aA ; n Table T~l.d,~ from t+-tyrosine in 1. These properties identify the 2-401 40 pigment as a melanin.

ii !!~iii~

4.6. Serology The molecular weight of mushroom ~oom tyrosinase, which occurs as four isozymic forms, ms, ranges from 26 to 120 kDa [20]. The associated :d enzyme (row 120000) is known to consist of two> heaw heavy and two light subunits with molecular wei:ights of 43000 and 13400, respectively. The molecul cular weights of the major components of the comm, )mmercially obtained mushroom tyrosinase were 55 000; 53 700;

14.31~ Fig. 1. Immunoblot of 2-40 cell lysate reacted against mushroomtyro~nase pol~lonal antibodies. Lane 1, 32 - 4 0 cell extract:lane 2, mushroomtyrosinase.Valuesindicate the positions of the molecularweightmarkers.

279

have been

igrifaciens: 500

and

0 w o u l d be ~e. Eucary90 k D a in tyrosinase [20],

DWLEDGEMENTS w o r k w a s f u n d e d by the M a r y l a n d Deat of N a t u r a l R e s o u r c e s contract g S-124by the M a r y l a n d Sea G r a n t College f u n d e d Slationai O c e a m c a n d A t m o s p h e r i c A d m i n a :~ Na88-AAD-00014, a n d by the Office of University Research Initiative T r a i n e e s h i p :g N-000-14-86-K = 0696, to W.C.F. a n d We apnreciate helpf, d discussion and .he )n o f strain 2 - 4 0 by G. Andrykovitch.

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[17] Laemmli, U.K. (1970) ! I [18] Towbin, l" H., Staehelin,

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Natl.I. Aca¢ Acad. Sci. U.S.A. [19] Ma~'un,o, K. and Wai( ~iochim. Biophys. Acta 872, 98-103. [20] Lerch. K (1981) in i~ zolOg/cal systems. (Sigel, H. and Sigel. A -146. Marcel Dekker. Inc.. New York a Sjoblad. !] [21] R.D. and Boll in Soil biochcmistry.,. Vol. 5. : (Paul, E.A. and Ladd, J.N., ¢ds.). pp.). 113-152, 1 Marcel Dekker. Inc., New York and Basel. [22] Kahn, V. and Andrawis. A. (1986) Phytochem hytochemistry 25, 333-337. [231 Andrawis, A. and Kahn, V. (19851 Phytochem lytochemistry 24, 397-405. [241 Huber, M. and Lerch, K. (19871 FEBS LetL 219. 335-338. " I C. and Triplett, E.L. (19851 J. Biol. Bio Chem. [25l Wittenber'g. 260, 12542-12546.