Recombinant Tyrosinase from Polyporus arcularius - ACS Publications

Subscriber access provided by - Access paid by the | UCSB Libraries

Article

Recombinant Tyrosinase from Polyporus arcularius: Overproduction in Escherichia coli, Characterization and Use in a Study of Aurones as Tyrosinase Effectors Eva Marková, Michael Kotik, Alena Krenkova, Petr Man, Romain Haudecoeur, Ahcene Boumendjel, Renaud Hardre, Yasmina Mekmouche, Elise Courvoisier-Dezord, Marius Réglier, and Ludmila Martinkova J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00286 • Publication Date (Web): 10 Mar 2016 Downloaded from http://pubs.acs.org on March 16, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32

Journal of Agricultural and Food Chemistry

1

Recombinant Tyrosinase from Polyporus arcularius: Overproduction in

2

Escherichia coli, Characterization and Use in a Study of Aurones as

3

Tyrosinase Effectors

4

Eva Marková†,‡, Michael Kotik†, Alena Křenková†, Petr Man†, Romain Haudecoeur&, Ahcène

5

Boumendjel&, Renaud Hardré#, Yasmina Mekmouche#, Elise Courvoisier-Dezord#, Marius

6

Réglier#, Ludmila Martínková†*

7

†

8

Czech Republic

9

‡

Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague,

Department of Biochemistry and Microbiology, Faculty of Food and Biochemical

10

Technology, University of Chemistry and Technology Prague, Technická 3, 166 28 Prague,

11

Czech Republic

12

&

13

#

14

France

Université Grenoble Alpes, CNRS, DPM UMR 5063, 38041, Grenoble, France

Aix Marseille Université, Centrale Marseille, CNRS, ISm2 UMR 7313, 13397, Marseille,

15 16

*Corresponding

17

[email protected]

author.

Phone:

420-29644-2569.

Fax:

420-29644-2509.

E-mail:

18

ACS Paragon Plus Environment

1


Page 2 of 32

19

Abstract: Tyrosinases act in development of organoleptic properties of tea, raisins etc., but

20

also cause unwanted browning of fruits, vegetables and mushrooms. The tyrosinase from

21

Agaricus bisporus has been used as a model to study tyrosinase inhibitors which are also

22

indispensable in the treatment of skin pigmentation disorders. However, this model has

23

disadvantages such as side enzyme activities and presence of multiple isoenzymes. Therefore,

24

we aimed to introduce a new tyrosinase model. The pro-tyrosinase from Polyporus arcularius

25

was overproduced in Escherichia coli. Trypsin digestion led to a cleavage after R388 and

26

hence enzyme activation. The tyrosinase was a homodimer and transformed L-DOPA and

27

tert-butylcatechol preferentially. Various aurons were examined as effectors of this enzyme.

28

2’- and 3’-Hydroxyaurones acted as its activators and 2’,4’-dihydroxyaurone as an inhibitor,

29

while 4’-hydroxyaurones were its substrates. The enzyme is a promising model for tyrosinase

30

effector studies, being a single isoenzyme and void of side enzyme activities.

31

Keywords: tyrosinase, Polyporus arcularius, Escherichia coli, trypsin digestion, aurones

32


2

Page 3 of 32


33

INTRODUCTION

34

Tyrosinases (EC 1.14.18.1) are dinuclear copper oxidases acting on phenolic compounds.

35

They catalyze the monooxygenation of monophenols, which is referred to as phenolase or

36

monooxygenase activity, and the oxidation of 1,2-diphenols (catechols), which is catecholase

37

or oxidase activity.1 Tyrosinases attract much attention because of their roles in skin

38

pigmentation and pigmentation disorders in mammals and in browning of some agricultural

39

products. The latter processes are desirable in fermented tea, cocoa or raisins, but unwanted in

40

some fruits, vegetables and mushrooms, where they occur during senescence or their

41

inappropriate handling after harvest.2 Thus a number of studies focused on tyrosinase

42

inhibitors as antibrowning agents for food industry and as drugs and cosmetics additives for

43

the treatment of hyperpigmentation.2-4 Tyrosinase applications in biodegradation,5-8

44

biotransformation,9,10 biopolymer grafting and cross-linking11-15 and in biosensors16-17 were

45

also examined. The uses of fungal and bacterial tyrosinases in these areas were reviewed

46

previously.18-20

47

The tyrosinase from Agaricus bisporus (common button mushroom) is readily available at

48

a low cost and has been thus used in the majority of the aforementioned studies. However, the

49

testing of potential tyrosinase inhibitors and antibrowning agents is not fully reliable with this

50

model, as it may contain several isoenzymes in differing ratios and various contaminants

51

(glycosidases, laccases, peroxidases, lectins, phenolic compounds).20 For this purpose,

52

recombinant fungal tyrosinases containing single isoenzymes and void of these contaminants

53

will therefore be more promising.

54

Plenty of putative tyrosinases and tyrosinase-like enzymes are encoded in fungal

55

genomes21 but few of them were studied at the protein level.21-24 Effective heterologous

56

expression systems are necessary to exploit this resource. One option is the production of the

57

latent tyrosinase form, its purification and its subsequent activation. Possible toxic effects of


3


Page 4 of 32

58

the active enzyme on the host, as well as browning or protein precipitation during the enzyme

59

purification can be thus avoided.25 In this way, the tyrosinases from Aspergillus oryzae and

60

Pholiota microspora (previously named Pholiota nameko) were produced in E. coli,26,27 but

61

the activity yields were not reported.

62

The aim of this work was to introduce a new model of a eukaryotic tyrosinase for

63

tyrosinase effector studies. To this end, the tyrosinase from the edible mushroom Polyporus

64

arcularius was selected. The corresponding gene and its light-regulated expression was

65

characterized in the fungus,28 but the properties of the protein product were unknown. Here

66

the protein was overproduced in E. coli, characterized and used to study the effects of natural

67

flavonoid aurones (benzylidenebenzofuran-3(2H)-one) and their derivatives on the tyrosinase

68

activity. Their effects were also previously tested with the tyrosinases from A. bisporus and

69

Streptomyces antibioticus. In those studies, some of them proved to be powerful tyrosinase

70

inhibitors, but others were tyrosinase activators or substrates, depending on their

71

hydroxylation pattern.29,30 Here, the previously described effects of aurones were compared

72

with those on the new tyrosinase from P. arcularius.

73 74 75 76

MATERIALS AND METHODS Chemicals. Aurones (Figure 1) were prepared as described previously.29,30 Other chemicals were purchased from standard sources.

77

Sequence analyses and DNA manipulations. Database searches and sequence

78

alignments were made using the programs BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi)

79

and Clustal W2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/), respectively. The synthetic gene

80

coding the pro-tyrosinase from P. arcularius HHB13444 (UniProt accession no. Q65Z70)

81

was designed in two parts without codon optimization, and purchased from Generay Biotech

82

Co. (Shanghai, China) and GenScript USA Inc., respectively, as pET28a-based constructs.


4

Page 5 of 32


83

The major N-terminal part of the gene encoding a 429-residue protein with an N-terminal His-

84

tag (1179 bp, its Kpn I restriction site eliminated) was obtained in pET28a under the control

85

of the T7 promoter (termed pET28ParcN). The C-terminal domain-encoding part of the gene

86

(702 bp) was received on a separate plasmid (pParcC) and was PCR-amplified using a

87

PfuUltra II Fusion HS DNA polymerase (Agilent Technologies), the primers ParcC-F (5′-

88

CCACTTGCCGTCAATCTGGCAAGCG-3′)

89

GATGGTACCCTACTAGGTAACCTGAAGGGAAGCAACGTG-3′, the Kpn I restriction

90

site being underlined), and pParcC as a template. Linearization of pET28ParcN was

91

accomplished by PCR using a Phusion DNA polymerase (Thermo Scientific), and the primers

92

ParcVec-F

93

ParcVec-R

94

G-3′, the Kpn I restriction site being underlined). The PCR products were gel-purified, 5′-

95

phosphorylated with T4 polynucleotide kinase (New England BioLabs), and restricted with

96

Kpn I. After purification using a High Pure PCR Product purification kit (Roche), the

97

restricted PCR products were ligated using a DNA ligation kit (TaKaRa Bio Inc.). The

98

ligation mixture was subsequently transformed into E. coli TOP10 cells (Life Technologies).

99

Plasmids from eight colonies were isolated, and the correct gene assembly was verified by

100

restriction analysis. The correctly assembled pro-tyrosinase-encoding gene in pET28a was

101

fully sequenced. The resulting plasmid carrying the gene coding for the pro-tyrosinase from

102

P. arcularius was designated pET28ParcP.

and

ParcC-R

(5′-GAACGCGGATCGCGCCCGGGAGCCGCCATAGAACTTG-3′)

(5′-

and

(5′-GTTGGTACCGCGGCCGCACTCGAGCACCACCACCACCACCACTGA

103

Gene expression. Competent E. coli BL21(DE3) cells (Life Technologies) were co-

104

transformed with plasmid pET28ParcP and pGro7, which is included in the Chaperone

105

Plasmid kit from Takara Bio Inc. The cells were grown in 500-mL Erlenmeyer flasks

106

containing 100 mL of LB medium supplemented with 0.2 mM CuSO4, kanamycin (50 µg

107

mL-1) and chloramphenicol (34 µg mL-1) for 3 h at 37 °C with shaking (220 rpm). Inducers of


5


Page 6 of 32

108

the expression of tyrosinase and GroEL/ES chaperones (0.02 mM isopropyl-β-D-1-

109

thiogalactopyranoside (IPTG) and 11.3 mM L-arabinose, respectively) were then added and

110

the cultivation temperature reduced to 20 °C. After a further 24-h cultivation, the cells were

111

harvested by centrifugation (30 min, 4 °C) and stored at -80 °C until use.

112

Enzyme extraction and affinity chromatography. The harvested biomass was

113

resuspended in binding buffer (20 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole,

114

pH 7.4). Lysozyme and DNAse were added to final concentrations of 200 and 20 µg mL-1,

115

respectively, and MgCl2 and PMSF to 1 mM each. The resulting slurry was incubated on ice

116

for 30 min and then sonicated on ice (Ultrasonic Homogenizer 4710 Series, Cole – Parmer,

117

USA; power output at 40%, 15 x 1 min). The crude extract was centrifuged (16,800 g, 20 min,

118

4 °C) and analyzed by SDS-PAGE. The supernatant was loaded onto 1-mL HisTrap FF

119

column (GE Healthcare). The pro-tyrosinase was eluted (1 mL min-1) with a linear gradient

120

(30 min) of 20-500 mM imidazole in 20 mM sodium phosphate, 500 mM NaCl, pH 7.4.

121

Fractions containing pro-tyrosinase were identified by SDS-PAGE and pooled.

122

Proteolytic digestion. The reaction mixture (total volume 1 mL) contained ca. 1 mg of

123

the pro-tyrosinase purified by affinity chromatography, 0.1 mg of trypsin (Sigma) and the

124

elution buffer (see above). The reaction proceeded at 25 °C with shaking (Thermomixer

125

Eppendorf; 800 rpm) and was terminated after 15 min by adding 2 mM PMSF. Alternatively,

126

trypsin was replaced with α-chymotrypsin (0.1 mg; Sigma) and the reaction was carried out

127

under the same conditions.

128

Size-exclusion chromatography. The molecular mass of the tyrosinase was assessed by

129

size-exclusion chromatography using a Superdex 200 10/300 GL column (GE Healthcare)

130

with a flow rate of 0.4 mL min-1. The mobile phase was 50 mM Tris/HCl, 150 mM NaCl (pH

131

7.2). The calibration proteins were cytochrome c, carbonic anhydrase, albumin, alcohol

132

dehydrogenase and β-amylase.


6

Page 7 of 32


133

Spectrophotometric activity assays. Tyrosinase activities for L-tyrosine, L-DOPA, TBC

134

and p-cresol were determined according to previously described methods (slightly

135

modified).31-33 The reaction mixtures (total volume 1 mL) contained 1 mM substrate, an

136

appropriate amount of the protein and 100 mM citrate buffer (pH 5.5). The reactions

137

proceeded at 50 °C for up to 10 min without shaking. The reaction products of TBC and p-

138

cresol were quantified at 400 nm (ε400 nm = 1,200 M-1 cm-1 and 1,433 M-1 cm-1, respectively)

139

and those of L-tyrosine and L-DOPA at 475 nm (ε475 nm = 3,400 M-1 cm-1).21,32,34 The increase

140

in absorbance was monitored continuously and the activities were calculated from the linear

141

part of the reaction. The pH and temperature optima and temperature stability were

142

determined in the same way using L-DOPA as the substrate. Enzyme kinetics were

143

determined in microtitration plates (reaction volume 0.2 mL) at pH 6.0 (phosphate buffer) and

144

30 °C using 0.02 – 2 mM of L-DOPA. The same method was used to determine the effects of

145

aurones (0.003 − 0.5 mM) on the enzyme activity for L-DOPA (1 mM) in the presence of 1 %

146

(v/v) dimethylsulfoxide. The inhibition of the enzyme by compound 1b was determined with

147

15.625-2000 µM - 2 mM of L-DOPA. The activities of the tyrosinase for its aurone substrates

148

2b and 3c were determined in the same way but without L-DOPA, and the decrease in

149

substrate concentrations was quantified at 400 nm as described previously.30

150

Activity assay by HPLC. Tyrosinase activity for phenol was determined in reaction

151

mixtures containing 1 mM of the substrate, 100 mM citrate buffer (pH 5.5), and an

152

appropriate amount of protein. The reactions proceeded at 50 °C for 2 - 20 min with shaking

153

(thermomixer; 800 rpm) and were terminated by adding 0.2 M HCl. After centrifuging the

154

samples, the concentration of phenol in the supernatants was determined by HPLC (column

155

Chromolith Speedrod RP18 (Merck), mobile phase 20% acetonitrile with 0.1% H3PO4, flow

156

rate 2 mL min-1). Activities for o-, and m-cresol were examined in an analogous way.


7


Page 8 of 32

157

Mass spectrometry. The protein was cut out of the SDS polyacrylamide gel, destained

158

and reduced (30 mM Tris-(2-carboxyethyl)phosphine, 90°C, 30 min). After alkylation of

159

cysteines with 30 mM iodoacetamide for 45 min and digestion with trypsin (Trypsin Gold,

160

Promega; 37°C), the peptides thus obtained were analyzed using an ApexQe 9.4T instrument

161

with MALDI source (Bruker Daltonics). MMass software (www.mmass.org)35 was used to

162

interpret the spectra. Intact monoisotopic mass of the protein was obtained by direct infusion

163

to ESI-FT-ICR MS after protein desalting on a protein microtrap (Optimize Technologies).

164

Data were processed in DataAnalysis 4.1 (Bruker Daltonics). Alternatively, the protein was

165

treated with 30 mM TCEP for 10 min at 75 °C prior to desalting. Fragmentation of the protein

166

was done using collision-induced dissociation in the quadrupole. For a detailed sequence

167

characterization and localization of modifications, the protein was analyzed using the system

168

described elsewhere.36 Briefly, the protein was dissolved in 0.5 M glycine HCl buffer pH 2.3

169

containing 4 M urea, and injected into an HPLC system consisting of an immobilized pepsin

170

column, reversed-phase trap column and analytical column connected to the ESI source of

171

FT-ICR MS. Data were acquired in LC-MS/MS mode and tandem mass spectra were

172

searched using the MASCOT algorithm against the sequence of the tyrosinase.

173 174

RESULTS

175

Pro-tyrosinase overproduction and partial purification by affinity chromatography.

176

The rationale behind designing the pro-tyrosinase gene in two parts (see Materials and

177

methods) was to compare the expression of the genes coding the entire pro-tyrosinase on one

178

hand, and only its active part (lacking the C-terminal region) on the other. However, no

179

activity was detected in the latter option, although a protein with an apparent molecular

180

weight of ca. 45 kDa (close to the expected ca. 47 kDa) was produced (Supporting

181

information; Figure S1). The codon frequency was not optimized to avoid translation rate


8

Page 9 of 32


182

being too high which may lead to misfolding.37 Expression of the total pro-tyrosinase gene

183

also gave a protein of the expected size (ca. 68 kDa). Variations in cultivation temperature

184

(15, 20 or 25 °C after induction) and in IPTG and CuSO4 concentrations (both 0.02 mM, 0.2

185

mM or 1 mM) were examined to increase the yield of the soluble enzyme further (data not

186

shown). Each parameter optimum found was used in subsequent testing. As a result, the

187

highest amount of the soluble protein was obtained with 0.02 mM IPTG, 0.2 mM CuSO4, at a

188

cultivation temperature of 20 °C. The pro-tyrosinase was partially purified by Ni affinity

189

chromatography (Figure 2, lane 3) of the cell extracts to give ca. 54 mg of protein L-1 of the

190

culture medium. This protein sample also contained the chaperones, which were produced by

191

this expression system and co-purified with the pro-tyrosinase as indicated by SDS-PAGE

192

showing a band corresponding in size to the GroEL subunit (ca. 60 kDa; Figure 2, lane 2-3).

193

Tyrosinase activation and gel filtration. A subsequent treatment of the partially purified

194

pro-tyrosinase by trypsin or α-chymotrypsin gave active tyrosinase. A specific activity ca.

195

25% higher was obtained with trypsin (Fig. S2). The trypsin digestion was therefore used in

196

the subsequent work. This also led to a marked reduction in the molecular size of the protein

197

(from ca. 68 kDa to ca. 43 kDa; Figure 2, lane 4). The enzyme obtained in this way lost its

198

ability to bind to the Ni affinity column, suggesting cleavage of the His-tag. At the same time,

199

the band of GroEL almost disappeared in the electrophoreogram (Figure 2, lanes 4-5). GroEL

200

contains multiple target sites for trypsin and it was thus probably digested into low-molecular-

201

weight peptides. The active tyrosinase was separated by size-exclusion chromatography,

202

which confirmed that it was the prevailing protein species in this sample. One of the minor

203

protein components probably corresponded to trypsin, as judged from its molecular weight of

204

ca. 23 kDa (Supporting information; Figure S2). The amount of the protein thus obtained was

205

ca. 30 mg of protein L-1 of the culture medium. The activity yield was ca. 3,800 U L-1 of the

206

culture medium for L-DOPA as the substrate (9,000 U L-1 for TBC). The molecular mass of


9


Page 10 of 32

207

the active tyrosinase was determined by size-exclusion chromatography to be 79.4 kDa,

208

which was in relatively close agreement with the molecular mass of a dimer of two N-

209

terminal fragments devoid of their His-tags (~86 kDa).

210

Mass spectrum analyses of the purified tyrosinase. Further examination of the protein

211

was done by peptide mapping, and revealed that the trypsin-catalyzed cleavage probably

212

produced a protein product delimited by sequences GSEFMSH- at the N-terminus and -

213

KRYGGSR at the C-terminus. The final product is thus most likely composed of 392 aa

214

residues and ends with R388. The recombinant protein probably also contained four aa

215

residues from the region following the N-terminal His6-tag, and was thus composed of 392 aa

216

residues (Supporting information; Figure S3). The two copper-coordinating sites were

217

predicted, which consist of three His-residues each and are conserved among tyrosinases

218

(ibid.). The molecular weight calculated for this protein (ca. 43.1 kDa) corresponded to that

219

determined by SDS-PAGE (ca. 43 kDa; Figure 2). To further confirm these findings, the

220

protein was subjected to intact mass measurement using FT-ICR MS. The measured

221

monoisotopic mass of 43090.025 Da was close to the expected calculated value for the active

222

tyrosinase (43092.116) but the mass error of 2 Da was too high for FT-MS measurement

223

(typically, accuracies in the ppm range are obtained). Since the protein reduction using TCEP

224

had no effect, we ruled out the presence of a disulphide bond. In addition, the MS/MS

225

analysis of the intact protein verified that the activated recombinant tyrosinase starts with the

226

sequence GSEFMSH- and ends with -KFYGGSR and that there are no modifications in the

227

71 N-terminal and 70 C-terminal amino acids. Therefore we considered the presence of a Cys-

228

His thioether bond, previously described in tyrosinases,38,39 as a possible explanation. Next,

229

we attempted to find experimental proof of this hypothesis. The protein was injected into an

230

immobilized pepsin column and the resulting peptides separated by a reversed-phase HPLC


10

Page 11 of 32


231

with subsequent MS/MS analysis. Using this approach, we localized the thioether connection

232

between C85 and H87 present in the sequence -GYCTHG-.

233

Characterization of the purified tyrosinase. The substrate range of the purified

234

tyrosinase was determined at a fixed substrate concentration (1 mM). The enzyme accepted

235

TBC, L-DOPA, p-cresol, phenol and L-tyrosine as its substrates with the relative activities of

236

100%, 42%, 37%, 4% and 1%, respectively; o- and m-cresol were not substrates. The relative

237

activities must be evaluated with some caution, the substrate concentrations not being

238

optimized. The Km was then determined for L-DOPA and shown to be relatively high (1.04 ±

239

0.08 mM). The enzyme exhibited kcat of 223 ± 8 s-1 for this substrate (Table 2; Supporting

240

information; Figure S4). The enzyme was active between pH 4 and 9, and exhibited more

241

than 60% of its maximum activity (found at pH 5.5) between pH 5 and 6 (Supporting

242

information; Figure S5). The enzyme exhibited its optimum activity at 50 °C, and retained 65

243

% of its maximum activity at 70 °C. It was unstable at temperatures over 55 °C (Supporting

244

information; Figure S6).

245

Effects of aurones on tyrosinase activity. Several aurones, some of which were

246

previously shown to differ in their behaviour towards the fungal tyrosinase from A. bisporus

247

and the bacterial tyrosinase from Streptomyces antiobioticus,30 were used for testing with the

248

tyrosinase from P. arcularius. In the previous study, compound 1b with a 2,4-disubstituted B-

249

ring (i.e. with a resorcinol moiety) was found to be a strong inhibitor of both fungal and

250

bacterial tyrosinase. Compounds 2a and 3b with a monosubstituted B-ring (substituted in

251

ortho- or meta-positions) were inhibitors of the bacterial enzyme, but activators of the fungal

252

one. Compounds 2b and 3c containing a para-substituted B-ring were substrates of both

253

enzymes, and compounds 1a and 3a with an unsubstituted B-ring did not exhibit any effect on

254

the enzymes. Both fungal tyrosinases (from A. bisporus and P. arcularius) exhibited the same

255

type of behaviour towards all the previously tested compounds, but differed in their Km or


11


Page 12 of 32

256

IC50 towards each of their substrates or inhibitors, respectively (Table 1; Supporting

257

information; Figures S7-S10). The values of both of these constants were generally higher for

258

the tyrosinase from P. arcularius, while those of the bacterial tyrosinase were the lowest

259

(except for compound 2b). The inhibition data of compound 1b were analyzed with the

260

program EZ-FIT.40 Based on the criteria describing the goodness-of-fit (standard errors,

261

Student t test, identifications of outlining data, and Akaike information criterion test), the

262

inhibition data best fitted the mixed inhibition model (Ki1 = 7 ± 1 µM, Ki2 = 27 ± 12 µM)

263

(Supporting information; Figures S11-12).

264 265

DISCUSSION

266

Recombinant tyrosinases from bacteria6,16,41-43, fungi21,23,24,26,27,33,44,45 and humans46,47 were

267

previously produced in differing yields and purities. In the fungal tyrosinases, C-terminal

268

proteolytic processing is required for its activity.48 The strategies for the expression of fungal

269

tyrosinase genes used either Pichia pastoris23 or some non-standard hosts such as Aspergillus

270

niger,45 Saccharomyces cerevisiae33 or Trichoderma reesei,23 which produced the enzymes in

271

their active forms. However, significant yields per 1 L of cell culture were only reported for

272

P. pastoris (ca. 24 mg of protein and 1,200 U) and A. niger (ca. 20 mg of protein and 1,668

273

U) producing the tyrosinases from T. reesei and P. sanguineus, respectively.24,45

274

In this work, a tyrosinase was produced in its latent form in E. coli and activated in vitro.

275

Previously, a pro-tyrosinase from P. microspora was produced in this host as a thioredoxin

276

fusion protein to improve its solubility,27 but the protein and activity yields have not been

277

reported. In our study, the yield (ca. 30 mg of the activated enzyme and ca. 3,800 U per 1 L of

278

the culture medium) was ca. 2-3 fold higher than for the two tyrosinases expressed in A. niger

279

or P. pastoris (see above). Thus the new tyrosinase was obtained in sufficient amounts to


12

Page 13 of 32


280

determine its substrate specificity and to use it as a model for testing potential tyrosinase

281

effectors.

282

The activation process, which removed the active site protection48,49, was studied in both

283

wild-type and recombinant producers of tyrosinases (Table S1). In almost all of these

284

enzymes, the proteolytic cleavage was found in the linker region between the C- and N-

285

terminal domains (ca. 26-31 aa residues downstream of the conserved Y(F)-X-Y motif21;

286

supplementary Figure S13). For instance, the C-terminal aa residue was identified in the

287

crystallized PPO3 and PPO4 tyrosinases from A. bisporus to be G392 and S383,

288

respectively.38,39 The C-terminal aa residue of the purified active tyrosinase in N. crassa was

289

F408.50 The proteolytic cleavage of the fungal pro-tyrosinases also proceeded in their

290

recombinant eukaryotic hosts in the corresponding regions.23,24,44,45 However, the tyrosinase

291

from Trichoderma reesei was cleaved at a different site when produced in Pichia pastoris

292

(R407) compared to the native host (G400).24 This could be one of the reasons for their

293

different pH profiles and slightly different specific activities. E. coli produced the latent forms

294

of tyrosinases, and it was thus necessary to cleave the enzymes in vitro. The enzyme from P.

295

microspora was probably processed after F387 by α-chymotrypsin, i.e. at the same site as the

296

endogenous tyrosinase.27 In the tyrosinase from P. arcularius, the cleavage site was predicted

297

to be F393 on the basis of the sequence alignment with the aforementioned enzymes

298

(Supporting information; Figure S13). The cleavage site in the recombinant enzyme digested

299

by trypsin was R388 and was thus close to the predicted one. The action of trypsin on the

300

evolutionarily distant tyrosinase from A. oryzae was different: the proenzyme was cleaved at

301

two sites (K312 and K457) and the holoenzyme thus consisted of two large and two small

302

subunits containing residues 2-312 and 313-457, respectively.26

303

It was speculated that the C-terminal domain may have similar effects to the chaperone-

304

like bacterial proteins which are probably responsible for copper incorporation into tyrosinase


13


Page 14 of 32

305

molecules.27 The notion of its importance for the post-translational processing of the pro-

306

tyrosinase was supported by the observation that the expression of genes coding for mature

307

tyrosinases from P. microspora, A. oryzae and A. bisporus failed to produce active enzymes.

308

26,27,51

309

conclusion.

Our attempts to express the tyrosinase from P. arcularius in this way led to the same

310

Few fungal tyrosinases, which share differing levels of amino acid sequence identities (29

311

- 69 %) with the new tyrosinase from P. arcularius, have been biochemically characterized so

312

far (Table 2). The majority of these enzymes exhibited apparent molecular weights of 42-45

313

kDa in SDS-PAGE. The number of subunits in the holoenzyme was determined in P.

314

sanguineus22 and P. arcularius, and the enzymes were found to be a monomer and a dimer,

315

respectively. However, it cannot be excluded that the dimer is artificially formed due to

316

protein cross-linking.25 The tyrosinase from A. oryzae, which is evolutionarily distant from

317

these enzymes (see above), is a heterotetramer consisting of two 36 kDa and two 18-kDa

318

subunits resulting from the double cleavage of the pro-tyrosinase.26 The endogenous

319

tyrosinases from A. bisporus are also composed of two large and two small subunits, the large

320

subunits being themselves active.25

321

The specific tyrosinase activity for L-DOPA was the highest in P. arcularius, followed by

322

P. sanguineus22, A. bisporus33 and T. resei23-24 (in this order). The Km values for L-DOPA were

323

in a broad range from approximately 1 µM in A. bisporus PPO2 isoenzyme33 or P. sanguineus44

324

to 1.930 mM in P. microspora,31 that of P. arcularius (1.04 mM) being similar to that in P.

325

microspora. The kcat for L-DOPA with the tyrosinase from P. arcularius was ca. half of that

326

with the tyrosinase from P. microspora, but higher than with the other fungal enzymes.

327

Tyrosinases generally exhibit lower activities for L-tyrosine compared to L-DOPA. For instance,

328

in the enzymes from T. reesei23,24 , A. bisporus47and the human tyrosinase47, the former

329

substrate was transformed at reaction rates one to two orders of magnitude lower. The


14

Page 15 of 32


330

tyrosinase from P. sanguineus22 and the bacterial tyrosinase from S. antibioticus47 exhibited

331

higher relative activites for L-tyrosine (ca. 25-30%) compared to L-DOPA. In the enzyme from

332

P. arcularius, this relative activity was only ca. 1%, which, however, could be also caused by

333

low substrate concentrations. It must be noted that using high concentrations of this substrate

334

(e.g. 10 mM) is difficult due to its low solubility in water (ca. 2.5 mM).23

335

The typical pH optimum of the enzymes was 6.0-7.0 but the tyrosinase from T. reesei

336

exhibited an optimum of pH 9.0 when expressed in the native organism. In contrast, the pH

337

optimum of the same enzyme produced in P. pastoris was lower (6.0),23,24 possibly due to

338

different proteolytic cleavage in different hosts and/or different glycosylation patterns. The pH

339

optimum of P. arcularius (5.5) was slightly lower than in the aforementioned enzymes.

340

Aurones is a type of flavonoids occurring in numerous plants, where they generate a gold-

341

yellow coloration. The therapeutic potential of these compounds and their synthetic analogues

342

have not yet been fully explored. One of their biological activities is their inhibition effect on

343

the tyrosinase activity in human melanocytes and the lead structure with B-ring hydroxylation

344

at the C-4´ position was identified. The inhibitory effects of such compounds seemed to be

345

much stronger than those of the well-known tyrosinase inhibitors arbutin and kojic acid. In

346

addition, toxicity studies in animals (rats, rabbits) revealed no adverse effects of these

347

aurones.52 The relationships between the substitution patterns and the inhibitory activity of

348

aurones was then also studied with the purified enzymes from A. bisporus, S. antibioticus30

349

and P. arcularius (this work) and the structures were optimized. Thus aurons with the

350

resorcinol moiety were the most powerful inhibitors of all three enzymes. In contrast, some

351

aurone analogues with other substitution patterns exhibited different effects on different

352

tyrosinases. It was previously hypothesized that this may be associated with the holoenzyme’s

353

structure (tetramer in A. bisporus, dimer in S. antibioticus). However, the P. arcularius

354

tyrosinase which probably is also a dimer, behaved in a similar way to the enzyme from A.


15


Page 16 of 32

355

bisporus. Thus the reason for the different behaviour of the fungal tyrosinases on one hand,

356

and bacterial on the other, may instead be the difference in the regions surrounding their

357

active sites.

358

In conclusion, a new tyrosinase from P. arcularius was produced at a high yield and its

359

utility as a model for studying tyrosinase inhibitors, activators and substrates was

360

demonstrated. An optimized E. coli expression system was used to produce the enzyme in its

361

latent form, which was then activated by a partial proteolytic cleavage. This strategy ruled out

362

the toxic effects of the active tyrosinase on the host as well as the unwanted manifestation of

363

the tyrosinase activity (potential protein oxidation and aggregation) during purification.

364

Compared to the commercial tyrosinase from A. bisporus, it has the advantage of having a

365

higher specific activity but, primarily, of containing only one isoenzyme and being void of the

366

impurities present in tyrosinases prepared from fungi.

367 368

ABBREVIATIONS USED

369

L-DOPA, 3,4-dihydroxy-L-phenylalanine

370

PMSF, phenylmethanesulfonyl fluoride

371

TBC, tert-butylcatechol

372

TCEP, tris(2-carboxyethyl)phosphine

373 374

ACKNOWLEDGEMENT

375

The Ministry of Education, Youth and Sports of the Czech Republic (project LD12049), the

376

Technology Agency of the Czech Republic (TA04021212), the Institute of Microbiology of

377

the Czech Academy of Sciences (internal project RVO61388971), the Agence Nationale pour

378

la Recherche (Labex Arcane ANR-11-LABX-0003-01 and Blanc program 2Cu-TargMelanin

379

ANR-09-BLAN-0028-01/02/03) and the European COST Program (COST action CM1003


16

Page 17 of 32


380

WG 2), within whose framework this work was carried out, are gratefully acknowledged for

381

their financial support. Access to the MS facility was enabled by the Operational Program

382

Prague – Competitiveness project (CZ.2.16/3.1.00/24023) and project LO1509 of the Ministry

383

of Education, Youth and Sports of the Czech Republic.

384

acknowledge the financial support of her short-term stay at the Aix Marseille Université

385

through the STSM-CM1003-300315-052174 fellowship.

386

The authors declare no competing financial interest.

E. Marková would like to

387 388

SUPPORTING INFORMATION

389

Table S1 Activation of wild-type and recombinant pro-tyrosinases by proteolytic cleavage.

390

Figure S1 SDS-PAGE of the cell extract from E. coli expressing the gene coding for the

391

hypothetical active part of tyrosinase from P. arcularius.

392

Figure S2 Purification of the trypsin-treated tyrosinase by size-exclusion chromatography.

393

Figure S3 Amino acid sequence of the His6-tagged pro-tyrosinase fusion protein.

394

Figure S4 Plot of initial rates of oxidation of L-DOPA catalysed by tyrosinase from P.

395

arcularius vs. substrate concentrations.

396

Figure S5 Effect of pH on the tyrosinase activity.

397

Figure S6 Effect of temperature on the tyrosinase activity (A) and stability (B).

398

Figure S7 Plots of initial rates of oxidation of aurones 2b (A) and 3c (B) catalysed by

399

tyrosinase from P. arcularius vs. substrate concentrations.

400

Figure S8 Effects of aurones 2a (A) and 3b (B) on L-DOPA oxidation catalysed by tyrosinase

401

from P. arcularius.


17


Page 18 of 32

402

Figure S9 Effect of aurone 1b on L-DOPA oxidation catalysed by tyrosinase from P.

403

arcularius.

404

Figure S10 Effects of aurones 1a (A) and 3a (B) on L-DOPA oxidation catalysed by

405

tyrosinase from P. arcularius.

406

Figure S11 Lineweaver-Burk plot of inhibition of tyrosinase from P. arcularius by aurone

407

1b.

408

Figure S12 Dixon plot of inhibition of tyrosinase from P. arcularius by aurone 1b.

409

Figure S13 Multiple sequence alignment of characterized fungal tyrosinases.

410

This material is available free of charge via the Internet at http://pubs.acs.org.

411


18

Page 19 of 32

412 413


REFERENCES (1)

Ramsden, C.A.; Riley, P.A. Tyrosinase: The four oxidation states of the active site and

414

their relevance to enzymatic activation, oxidation and inactivation. Bioorg. Med. Chem. 2014,

415

22, 2388-2395.

416 417 418 419 420 421 422 423 424 425 426

(2)

Seo, S.-Y.; Sharma, V.K.; Sharma, N. Mushroom tyrosinase: recent prospects. J.

Agric. Food Chem. 2003, 51, 2837–2853. (3)

Chang, T.S. An updated review of tyrosinase inhibitors. Int. J. Mol. Sci. 2009, 10,

2440-2475. (4)

Loizzo, M.R.; Tundis, R.; Menichini, F. Natural and synthetic tyrosinase inhibitors as

antibrowning agents: an update. Compr. Rev. Food. Sci. F. 2012, 11, 378-398. (5)

Faccio, G.; Kruus, K.; Saloheimo, M.; Thöny-Meyer, L. Bacterial tyrosinases and their

applications. Process. Biochem. 2012, 47, 1749–1760. (6)

Fairhead, M.; Thöny-Meyer, L. Bacterial tyrosinases: old enzymes with new relevance

to biotechnology. New Biotech. 2012, 29, 183–191. (7)

Mukherjee, S.; Basak, B.; Bhunia, D.A.; Mondal, B. Potential use of polyphenol

427

oxidases (PPO) in the bioremediation of phenolic contaminants containing industrial

428

wastewater. Rev. Environ. Sci. Bio-Technol. 2013, 12, 61-73.

429 430 431 432

(8)

Rauf, M.A., Ashraf, S.S. Survey of recent trend in biochemically assisted degradation

of dyes. Chem. Eng. J. 2012, 209, 520-530. (9)

Kües, U. Fungal enzymes for environmental management. Curr. Opin. Biotechnol.

2015, 33, 268-278.

433

(10) Martínková, L., Kotik, M., Marková, E., Homolka, L. Biodegradation of phenolic

434

compounds by Basidiomycota and its phenol oxidases: A review. Chemosphere 2016, 149,

435

373-382.


19


436 437

Page 20 of 32

(11) Xu, D.-Y., Chen, J.-Y., Yang, Z. Use of cross-linked tyrosinase aggregates as catalyst for synthesis of L –DOPA. Biochem. Eng. J. 2012, 63, 88-94.

438

(12) Botta, G.; Bizzarri, B.M.; Garozzo, A.; Timpanaro, R.; Bisignano, B.; Amatore, D.;

439

Palamara, A.T.; Nencioni, L.; Saladino, R. Carbon nanotubes supported tyrosinase in the

440

synthesis of lipophilic hydroxytyrosol and dihydrocaffeoyl catechols with antiviral activity

441

against DNA and RNA viruses. Bioorg. Med. Chem. 2015, 23, 5345-5351.

442

(13) Anghileri, A.; Lantto, R.; Kruus, K.; Arosio, C.; Freddi, G. Tyrosinase-catalyzed

443

grafting of sericin peptides onto chitosan and production of protein–polysaccharide

444

bioconjugates. J. Biotechnol. 2007, 127, 508–519.

445

(14) Jus, S.; Stachel, I.; Schloegl, W.; Pretzler, M.; Friess, W.; Meyer, M.; Birner-

446

Gruenberger, R.; Gübitz, G.M. Cross-linking of collagen with laccases and tyrosinases. Mater.

447

Sci. Eng. 2011, C31, 1068-1077.

448

(15) Lewandowski, A.T.; Small, D.A.; Chen, T.; Payne, G.F.; Bentley, W.E. Tyrosine-

449

based ‘‘activatable pro-tag’’: enzyme-catalyzed protein capture and release. Biotechnol.

450

Bioeng. 2006, 93, 1207–1215.

451

(16) Fairhead, M.; Thöny-Meyer, L. Cross-linking and immobilisation of different proteins

452

with recombinant Verrucomicrobium spinosum tyrosinase. J. Biotechnol. 2010, 150, 546–551.

453

(17) Faccio, G.; Kämpf, M.M.; Piatti, C.; Thöny-Meyer, L.; Richter, M. Tyrosinase-

454

catalyzed site-specific immobilization of engineered C-phycocyanin to surface. Sci. Rep.

455

2014, 4, art. no. 5370.

456 457 458 459

(18) Gu, B.X.; Xu, C.X.; Zhu, G.P.; Liu, S.Q.; Chen, L.Y.; Li, X.S. Tyrosinase immobilization on ZnO nanorods for phenol detection. J. Phys. Chem. B 2009, 113, 377–381. (19) Noh, S.; Yang, H. Sensitive phenol detection using tyrosinase-based phenol oxidation combined with redox cycling of catechol. Electroanalysis 2014, 26, 2727-2731.


20

Page 21 of 32


460

(20) Flurkey, A.; Cooksey, J.; Reddy, A.; Spoonmore, K.; Rescigno, A.; Inlow, J.; Flurkey,

461

W.H. Enzyme, protein, carbohydrate, and phenolic contaminants in commercial tyrosinase

462

preparations: potential problems affecting tyrosinase activity and inhibition studies. J. Agric.

463

Food Chem. 2008, 56, 4760–4768.

464

(21) Gasparetti, C.; Faccio, G.; Arvas, M.; Buchert, J.; Saloheimo, M.; Kruus, K. Discovery

465

of a new tyrosinase-like enzyme family lacking a C-terminally processed domain: production

466

and characterization of an Aspergillus oryzae catechol oxidase. Appl. Microbiol. Biotechnol.

467

2010, 86, 213-226.

468

(22) Halaouli, S.; Asther, M.; Kruus, K.; Guo, L.; Hamdi, M.; Sigoillot, J.-C.; Asther, M.;

469

Lomascolo, A. Characterization of a new tyrosinase from Pycnoporus species with high

470

potential for food technological applications. J. Appl. Microbiol. 2005, 98, 332-343.

471

(23) Selinheimo, E.; Saloheimo, M.; Ahola, E.; Westerholm-Parvinen, A.; Kalkkinen, N.;

472

Buchert, J.; Kruus, K. Production and characterization of a secreted, C-terminally processed

473

tyrosinase from the filamentous fungus Trichoderma reesei. FEBS J. 2006, 273, 4322–4335.

474

(24) Westerholm-Parvinen, A.; Selinheimo, E.; Boer, H.; Kalkkinen, N.; Mattinen, M.;

475

Saloheimo, M. Expression of the Trichoderma reesei tyrosinase 2 in Pichia pastoris: isotopic

476

labelling and physicochemical characterization. Protein Expres. Purif. 2007, 55, 147–158.

477

(25) Mauracher, S.G.; Molitor, C.; Michael, C.; Kragl, M.; Rizzi, A.; Rompel, A. High

478

level protein-purification allows the unambiguous polypeptide determination of latent isoform

479

PPO4 of mushroom tyrosinase. Phytochemistry 2014, 99, 14-25.

480

(26) Fujieda, N.; Murata, M.; Yabuta, S.; Ikeda, T.; Shimokawa, C.; Nakamura, Y.; Hata,

481

Y.; Itoh, S. Multifunctions of MelB, a fungal tyrosinase from Aspergillus oryzae.

482

ChemBioChem 2012, 13, 193–201.


21


Page 22 of 32

483

(27) Kawamura-Konishi, Y.; Maekawa, S., Tsuji, M.; Goto, H. C-terminal processing of

484

tyrosinase is responsible for activation of Pholiota microspora proenzyme. Appl. Microbiol.

485

Biotechnol. 2011, 90, 227-234.

486 487

(28) Kanda, S.; Aimi, T.; Masumoto, S.; Nakano, K.; Kitamoto, Y.; Morinaga, T. Photoregulated tyrosinase gene in Polyporus arcularius. Mycoscience 2007, 48, 34-41.

488

(29) Dubois, C.; Haudecoeur, R.; Orio, M.; Belle, C.; Bochot, C.; Boumendjel, A.; Hardré,

489

R.; Jamet, H.; Réglier, M.. Versatile effects of aurone structure on mushroom tyrosinase

490

activity. ChemBioChem 2012, 13, 559-565.

491

(30) Haudecoeur, R.; Gouron, A.; Dubois, C.; Jamet, H.; Lightbody, M.; Hardré, R.; Milet,

492

A.; Bergantino, E.; Bubacco, L.; Belle, C.; Réglier, M.; Boumendjel, A. Investigation of

493

binding-site homology between mushroom and bacterial tyrosinases by using aurones as

494

effectors. ChemBioChem 2014, 15, 1325-1333.

495

(31) Kawamura-Konishi, Y.; Tsuji, M.; Hatana, S.; Asanuma, M.; Kakuta, D.; Kawano, T.;

496

Mukouyama, E.B.; Goto, H.; Suzuki, H. Purification, characterization, and molecular cloning

497

of tyrosinase from Pholiota nameko. Biosci. Biotech. Bioch. 2011, 90, 227-234.

498 499

(32) Selinheimo, E.; Autio, K.; Kruus, K.; Buchert, J. Elucidating the mechanism of laccase and tyrosinase in wheat bread making. J. Agric. Food Chem. 2007, 55, 6357−6365.

500

(33) Lezzi, C.; Bleve, G.; Spagnolo, S.; Perrotta, C.; Grieco, F. Production of recombinant

501

Agaricus bisporus tyrosinase in Saccharomyces cerevisiae cells. J. Ind. Microbiol.

502

Biotechnol. 2012, 39, 1875-1880.

503

(34) Orenes-Piñero, E.; García-Carmona, F.; Sánchez-Ferrer, A. Kinetic characterization of

504

cresolase activity of Streptomyces antibioticus tyrosinase. Enzyme Microb. Technol. 2006, 39,

505

158-163.


22

Page 23 of 32


506

(35) Strohalm, M.; Hassman, M.; Košata, B.; Kodíček, M. mMass data miner: an open

507

source alternative for mass spectrometric data analysis. Rapid Commun. Mass Spectrom.

508

2008, 22, 905-908.

509

(36) Kadek, A.; Mrazek, H.; Halada, P.; Rey, M.; Schriemer, D.C.; Man, P. Aspartic

510

protease nepenthesin-1 as a tool for digestion in hydrogen/deuterium exchange mass

511

spectrometry. Anal. Chem. 2014, 86, 4287-4294.

512

(37) Petříčková, A.; Veselá, A.B.; Kaplan, O.; Kubáč, D.; Uhnáková, B.; Malandra, A.;

513

Felsberg, J.; Rinágelová, A.; Weyrauch, P.; Křen, V.; Bezouška, K.; Martínková, L.

514

Purification and characterization of heterologously expressed nitrilases from filamentous

515

fungi. Appl. Microbiol. Biotechnol. 2012, 93, 1553-1561.

516

(38) Ismaya, W.T.; Rozeboom, H.J.; Weijn, A.; Mes, J.J.; Fusetti, F.; Wichers, H.J.;

517

Dijkstra, B.W. Crystal structure of Agaricus bisporus mushroom tyrosinase: identity of the

518

tetramer subunits and interaction with tropolone. Biochemistry 2011, 50, 5477-5486.

519

(39) Mauracher, S.G.; Molitor, C.; Al-Oweini, R.; Kortz, U.; Rompel, A. Latent and active

520

abPPO4 mushroom tyrosinase cocrystallized with hexatungstotellurate(VI) in a single crystal.

521

Acta Crystallogr. Sect. D-Biol. Crystallogr. 2014, 70, 2301–2315.

522

(40) Perrella, F.W. EZ-FIT: A practical curve-fitting microcomputer program for the

523

analysis of enzyme kinetic data on IBM-PC compatible computers. Anal. Biochem. 1988, 174,

524

437-447.

525

(41) Ito, M.; Inouye, K. Catalytic properties of an organic solvent-resistant tyrosinase from

526

Streptomyces sp. REN-21 and its high-level production in E. coli. J. Biochem. 2005, 138,

527

355–362.

528

(42) Shuster, V.; Fishman, A. Isolation, cloning and characterization of a tyrosinase with

529

improved activity in organic solvents from Bacillus megaterium. J. Mol. Microbiol.

530

Biotechnol. 2009, 17, 188–200.


23


531 532

Page 24 of 32

(43) Ren, Q.; Henes, B.; Fairhead, M.; Thöny-Meyer, L. High level production of tyrosinase in recombinant Escherichia coli. BMC Biotechnol. 2013, 13, art. no. 18.

533

(44) Halaouli, S.; Asther, M.; Sigoillot, J.-C.; Hamdi, M.; Lomascolo, A. Fungal

534

tyrosinases: new prospects in molecular characteristics, bioengineering and biotechnological

535

applications. J. Appl. Microbiol. 2006, 100, 219–232.

536

(45) Halaouli, S.; Record, E.; Casalot, L.; Hamdi, M.; Sigoillot, J.-C.; Asther, M.;

537

Lomascolo, A. Cloning and characterization of a tyrosinase gene from the white-rot fungus

538

Pycnoporus sanguineus, and overproduction of the recombinant protein in Aspergillus niger.

539

Appl. Microbiol. Biotechnol. 2006, 70, 580-589.

540

(46) Dolinska, M.B.; Kovaleva, E.,; Backlund, P.,; Wingfield, P.T.,; Brooks, B.P.,;

541

Sergeev, Y.V. Albinism-causing mutations in recombinant human tyrosinase alter intrinsic

542

enzymatic activity. PLOS One 2014, 9, e84494.

543

(47) Fogal, S.; Carotti, M.; Giaretta, L., Lanciai, F., Nogara, L., Bubacco, L., Bergantino,

544

E. Human tyrosinase produced in insect cells: a landmark for the screening of new drugs

545

addressing its activity. Mol. Biotechnol. 2015, 57, 45-57.

546 547 548

(48) Flurkey, W.H.; Inlow, J.K. Proteolytic processing of polyphenol oxidase from plants and fungi. J. Inorg. Biochem. 2008, 102, 2160-2170. (49) Faccio, G.; Arvas, M.; Thöny-Meyer, L.; Saloheimo, M. Experimental and

549

bioinformatic investigation of the proteolytic degradation of the C-terminal domain of a

550

fungal tyrosinase. J. Inorg. Biochem. 2013, 121, 37-45.

551

(50) Kupper, U.; Niedermann, D.M.; Travaglini, G.; Lerch, K. Isolation and

552

characterization of the tyrosinase gene from Neurospora crassa. J. Biol. Chem. 1989, 264,

553

17250-17258.


24

Page 25 of 32


554

(51) Wu, J., Chen, H., Gao, J., Liu, X., Cheng, W., Ma, X. Cloning, characterization and

555

expression of two new polyphenol oxidase cDNAs from Agaricus bisporus. Biotechnol. Lett.

556

2010, 32, 1439–1447.

557

(52) Okombi, S., Rival, D., Bonnet, S., Mariotte, A.-M., Perrier, E., Boumendjel, A.

558

Discovery of benzylidenebenzofuran-3(2H)-one (aurones) as inhibitors of tyrosinase derived

559

from human melanocytes. J. Med. Chem. 2006, 49, 329-333.

560


25


Page 26 of 32

561

Figure 1. Structures of tyrosinase effectors examined in this work

562

Figure 2. Production in E. coli and partial purification of tyrosinase from Polyporus

563

arcularius. Lane 1: marker; Lane 2: cell-free extract; Lane 3: pro-tyrosinase purified by Ni

564

affinity chromatography; Lane 4: trypsin-treated tyrosinase; Lane 5: trypsin-treated

565

tyrosinase purified by size-exclusion chromatography. Molecular weight markers in kDa.

566

Pro-tyrosinase (68 kDa) and GroEL (60 kDa) in lane 3 and tyrosinase (43 kDa) in lane 5 are

567

indicated with arrows.


26

Page 27 of 32


Table 1. Activity of aurones towards tyrosinases from Agaricus bisporus, Streptomyces antiobioticus and Polyporus arcularius Tyrosinase from Compound

1a

1b

2a

2b

3a

3b

3c

Streptomyces

Polyporus arcularius

Agaricus bisporus

n.a.

n.a.

n.a.

I

I

I

(IC50 = 34 ± 3 µM)

(IC50 = 9 ± 1 µM)

(IC50 = 4 ± 1 µM)

H.A

H.A.

I

(145%)

(240%)

(IC50 > 1 mM)

S

S

S

(Km = 26 ± 9 µM)

(Km = 4.8 ± 0.1 µM)

(Km = 9 ± 2 µM)

n.a.

n.a.

n.a.

H.A

H.A.

I

(167%)

(150%)

(IC50 > 0.2 mM)

S

S

S

(Km = 63±20 µM )

(Km = 18.2±0.1 µM )

(Km = 0.20 ± 0.02 µM)

antiobioticus

H.A. = hyperbolic activation; I = inhibitor, S = substrate, n. a. = no activity; n.d. = not determined Note: The Km or IC50 (µM) towards each substrate or inhibitor, respectively, is given in brackets. Data for the tyrosinases from A. bisporus and S. antiobioticus are adopted from the previous work (Haudecoeur et al. 2014).


27


Page 28 of 32

Table 2. Biochemical properties of purified fungal tyrosinases Source

Identity, %

Molecular mass, kDa Specific activity,

Kinetic parameters,

(cover, %)a

-1 (Subunit/holoenzyme) U mg protein

Km, µM/kcat, s-1

(substrate)

Agaricus bisporus

Reference

(substrate)

40 (58)

65, 21/n.d.

27.4±3.4 (L-DOPA)

1.22/141 (L-DOPA)

(33)

Aspergillus oryzae

29 (50)

36, 18/102

n.d.

43/49 (L-tyrosine)

(26)

Pholiota microspora

55 (98)

42b, 44c/n.d.

412 (TBC)b

1,930/478 (L-DOPA)b

(27, 31)

(PPO2 isoform)

163/294 (TBC)b 189/162 (TBC)c Polyporus arcularius

-

43/79

127 ± 9 (L-DOPA)

1,040/223 (L-DOPA)

This work

84.3 (L-DOPA)

0.9/85 (L-DOPA)

(22)

30.2 (L-tyrosine)

1.0/41 (L-tyrosine)

303 ± 17 (TBC) Pycnoporus sanguineus

69 (98)

45/45

28 ACS Paragon Plus Environment

Page 29 of 32

Trichoderma reesei


35 (67)

43/n.d.

15.4-23.0 (L-DOPA)d

3,000/22 (L-DOPA)

(23, 24)

0.42-0.54 (L-tyrosine)d a

comparing the amino acid sequences to tyrosinase from P. arcularius (template)

b

endogenous

c

recombinant

d

different for tyrosinases produced in different hosts (T. reesei, P. pastoris)

29 ACS Paragon Plus Environment


R3 R4

B

R1

O R5

A R2

O

Page 30 of 32

1a R1, R3, R4, R5 = H; R2 = OH 1b R1, R4 = H; R2, R3; R5 = OH 2a R 2, R3, R5 = H; R1, R4 = OH 2b R2, R4, R5 = H; R1, R3 = OH 3a R3, R4, R5 = H; R1, R2 = OH 3b R3, R5 = H; R1, R2, R4 = OH 3c R4, R5 = H; R1, R2, R3 = OH

Figure 1


30

Page 31 of 32


kDa

1

2

3

4

5

97 66

45

30

21

14

Figure 2


31


TOC graphic 191x129mm (96 x 96 DPI)


Page 32 of 32

Recombinant Tyrosinase from Polyporus arcularius - ACS Publications

Recommend Documents