Biochemical and structural characterisation of the copper containing oxidoreductases catechol oxidase, tyrosinase, and laccase from ascomycete fungi: Dissertation

Chiara Gasparetti

Research output: ThesisDissertationCollection of Articles

Abstract

Catechol oxidase (EC 1.10.3.1), tyrosinase (EC 1.14.18.1), and laccase (EC 1.10.3.2) are copper-containing metalloenzymes. They oxidise substituted phenols and use molecular oxygen as a terminal electron acceptor. Catechol oxidases and tyrosinases catalyse the oxidation of p-substituted o-diphenols to the corresponding o-quinones. Tyrosinases also catalyse the introduction of a hydroxyl group in the ortho position of p-substituted monophenols and the subsequent oxidation to the corresponding o-quinones. Laccases can oxidise a wide range of compounds by removing single electrons from the reducing group of the substrate and generate free radicals. The reaction products of these oxidases can react further non-enzymatically and lead to formation of polymers and cross-linking of proteins and carbohydrates, in certain conditions. The work focused on examination of the properties of catechol oxidases, tyrosinases and laccases. A novel catechol oxidase from the ascomycete fungus Aspergillus oryzae was characterised from biochemical and structural point of view. Tyrosinases from Trichoderma reesei and Agaricus bisporus were examined in terms of substrate specificity and inhibition. The oxidation capacity of laccases was elucidated by using a set of laccases with different redox potential and a set of substituted phenolic substrates with different redox potential. Finally, an evaluation of the protein cross-linking ability of catechol oxidase from A. oryzae, tyrosinases from T. reesei and A. bisporus and laccases from Trametes hirsuta, Thielavia arenaria, and Melanocarpus albomyces was performed. A novel extracellular catechol oxidase from A. oryzae (AoCO4; UniProtKB: Q2UNF9; Entrez gene ID: 5990879) was chosen for cloning and expression as representative of the newly discovered family of short tyrosinases sequences. AoCO4 gene was heterologously expressed in T. reesei. The protein produced did not show activity on L-tyrosine and 3,4-dihydroxy-L-phenylalanine (L-DOPA), which are typical substrates for tyrosinases. Consequently, the protein was classified as a catechol oxidase. AoCO4 was produced in a bioreactor and the expression resulted in high yields. The purified AoCO4 was partially processed at a Kex2/furin-type protease site and showed a molecular weight of 39.3 kDa. AoCO4 was able to oxidise a limited range of diphenolic compounds, e.g., catechol, caffeic acid, hydrocaffeic acid, and 4-tert-buthylcatechol. AoCO4 oxidised also the monophenolic compounds aminophenol and guaiacol. AoCO4 showed a pH optimum in the acidic range and was observed to be a relatively thermostable enzyme. A crystal structure of AoCO4 was solved at 2.5 Å resolution. AoCO4 was a monomer and the overall structure of AoCO4 was found to be similar to the known structures of catechol oxidases and tyrosinases. A detailed characterisation of the substrate-specificity of the extracellular tyrosinase from T. reesei (TrT) was accomplished and compared to the commercial tyrosinase from A. bisporus (AbT). TrT generally showed lower affinity than AbT on substrates that had a free amino group, such as L-tyrosine, L-DOPA, and YGG tripeptide. The reaction end product of TrT and AbT was studied via mass spectrometry and dopachrome was found to be the only reaction end product of L-DOPA oxidation catalysed by both tyrosinases. Dopachrome produced by AbT-catalysed oxidation of L-DOPA was also shown to inhibit the TrT tyrosinase by an end product inhibition mechanism. Further, when the type of inhibition of potential inhibitors for TrT was analysed with p-coumaric acid and caffeic acid as substrates, potassium cyanide and kojic acid were the strongest inhibitors of TrT. The kinetics of three laccases, with different redox potential (E°), for three p-substituted dimethoxy phenolic substrates (2,6-dimethoxyphenol, syringic acid, and methyl syringate) with different E° were determined at two pHs. The laccases studied were from M. albomyces, T. arenaria, and T. hirsuta. The enzyme from the ascomycete fungus T. arenaria was a novel laccase produced in T. reesei. T. arenaria laccase was purified, and biochemically and structurally characterised. By comparison of the three laccases it could be shown that both the difference in redox potential (?E°) and the pH had an effect on the kinetics. However, the effect of ?E° was found to prevail over that of the pH for substrates with a high E°, such as methyl syringate. All oxidative enzymes studied in this work were also tested for their cross-linking ability, with ?-caseins utilised as a model substrate with and without a cross-linking agent. TrT was found to be the best enzyme for cross-linking of ?-caseins, whereas AbT was found to be the best enzyme for cross-linking ?-caseins in the presence of catechol as a cross-linking agent. Interestingly, AoCO4 was also found to cross-link ?-caseins in the presence of catechol as a cross-linking agent.
Original languageEnglish
QualificationDoctor Degree
Awarding Institution
  • Aalto University
Supervisors/Advisors
  • Kruus, Kristiina, Supervisor
  • Buchert, Johanna, Supervisor, External person
Award date26 Oct 2012
Place of PublicationEspoo
Publisher
Print ISBNs978-951-38-7934-1
Electronic ISBNs978-951-38-7935-8
Publication statusPublished - 2012
MoE publication typeG5 Doctoral dissertation (article)

Fingerprint

Catechol Oxidase
Laccase
Monophenol Monooxygenase
Fungi
Copper
Oxidoreductases
Substrates
Tyrosine
Caseins
Phenylalanine
Oxidation
Enzymes
Quinones
tyrosyl-glycyl-glycine
Proteins
Genes
Potassium Cyanide
Furin
Aminophenols
Guaiacol

Keywords

  • Trichoderma reesei
  • Agaricus bisporus
  • Aspergillus oryzae
  • Thielavia arenaria
  • tyrosinase
  • catechol oxidase
  • laccase
  • purification
  • characterisation
  • oxidation capacity
  • inhibition
  • three-dimensional structure
  • cross-linking

Cite this

@phdthesis{53b01ccfcafe44d0af88f22ce8f1bd13,
title = "Biochemical and structural characterisation of the copper containing oxidoreductases catechol oxidase, tyrosinase, and laccase from ascomycete fungi: Dissertation",
abstract = "Catechol oxidase (EC 1.10.3.1), tyrosinase (EC 1.14.18.1), and laccase (EC 1.10.3.2) are copper-containing metalloenzymes. They oxidise substituted phenols and use molecular oxygen as a terminal electron acceptor. Catechol oxidases and tyrosinases catalyse the oxidation of p-substituted o-diphenols to the corresponding o-quinones. Tyrosinases also catalyse the introduction of a hydroxyl group in the ortho position of p-substituted monophenols and the subsequent oxidation to the corresponding o-quinones. Laccases can oxidise a wide range of compounds by removing single electrons from the reducing group of the substrate and generate free radicals. The reaction products of these oxidases can react further non-enzymatically and lead to formation of polymers and cross-linking of proteins and carbohydrates, in certain conditions. The work focused on examination of the properties of catechol oxidases, tyrosinases and laccases. A novel catechol oxidase from the ascomycete fungus Aspergillus oryzae was characterised from biochemical and structural point of view. Tyrosinases from Trichoderma reesei and Agaricus bisporus were examined in terms of substrate specificity and inhibition. The oxidation capacity of laccases was elucidated by using a set of laccases with different redox potential and a set of substituted phenolic substrates with different redox potential. Finally, an evaluation of the protein cross-linking ability of catechol oxidase from A. oryzae, tyrosinases from T. reesei and A. bisporus and laccases from Trametes hirsuta, Thielavia arenaria, and Melanocarpus albomyces was performed. A novel extracellular catechol oxidase from A. oryzae (AoCO4; UniProtKB: Q2UNF9; Entrez gene ID: 5990879) was chosen for cloning and expression as representative of the newly discovered family of short tyrosinases sequences. AoCO4 gene was heterologously expressed in T. reesei. The protein produced did not show activity on L-tyrosine and 3,4-dihydroxy-L-phenylalanine (L-DOPA), which are typical substrates for tyrosinases. Consequently, the protein was classified as a catechol oxidase. AoCO4 was produced in a bioreactor and the expression resulted in high yields. The purified AoCO4 was partially processed at a Kex2/furin-type protease site and showed a molecular weight of 39.3 kDa. AoCO4 was able to oxidise a limited range of diphenolic compounds, e.g., catechol, caffeic acid, hydrocaffeic acid, and 4-tert-buthylcatechol. AoCO4 oxidised also the monophenolic compounds aminophenol and guaiacol. AoCO4 showed a pH optimum in the acidic range and was observed to be a relatively thermostable enzyme. A crystal structure of AoCO4 was solved at 2.5 {\AA} resolution. AoCO4 was a monomer and the overall structure of AoCO4 was found to be similar to the known structures of catechol oxidases and tyrosinases. A detailed characterisation of the substrate-specificity of the extracellular tyrosinase from T. reesei (TrT) was accomplished and compared to the commercial tyrosinase from A. bisporus (AbT). TrT generally showed lower affinity than AbT on substrates that had a free amino group, such as L-tyrosine, L-DOPA, and YGG tripeptide. The reaction end product of TrT and AbT was studied via mass spectrometry and dopachrome was found to be the only reaction end product of L-DOPA oxidation catalysed by both tyrosinases. Dopachrome produced by AbT-catalysed oxidation of L-DOPA was also shown to inhibit the TrT tyrosinase by an end product inhibition mechanism. Further, when the type of inhibition of potential inhibitors for TrT was analysed with p-coumaric acid and caffeic acid as substrates, potassium cyanide and kojic acid were the strongest inhibitors of TrT. The kinetics of three laccases, with different redox potential (E°), for three p-substituted dimethoxy phenolic substrates (2,6-dimethoxyphenol, syringic acid, and methyl syringate) with different E° were determined at two pHs. The laccases studied were from M. albomyces, T. arenaria, and T. hirsuta. The enzyme from the ascomycete fungus T. arenaria was a novel laccase produced in T. reesei. T. arenaria laccase was purified, and biochemically and structurally characterised. By comparison of the three laccases it could be shown that both the difference in redox potential (?E°) and the pH had an effect on the kinetics. However, the effect of ?E° was found to prevail over that of the pH for substrates with a high E°, such as methyl syringate. All oxidative enzymes studied in this work were also tested for their cross-linking ability, with ?-caseins utilised as a model substrate with and without a cross-linking agent. TrT was found to be the best enzyme for cross-linking of ?-caseins, whereas AbT was found to be the best enzyme for cross-linking ?-caseins in the presence of catechol as a cross-linking agent. Interestingly, AoCO4 was also found to cross-link ?-caseins in the presence of catechol as a cross-linking agent.",
keywords = "Trichoderma reesei, Agaricus bisporus, Aspergillus oryzae, Thielavia arenaria, tyrosinase, catechol oxidase, laccase, purification, characterisation, oxidation capacity, inhibition, three-dimensional structure, cross-linking",
author = "Chiara Gasparetti",
year = "2012",
language = "English",
isbn = "978-951-38-7934-1",
series = "VTT Science",
publisher = "VTT Technical Research Centre of Finland",
number = "16",
address = "Finland",
school = "Aalto University",

}

Biochemical and structural characterisation of the copper containing oxidoreductases catechol oxidase, tyrosinase, and laccase from ascomycete fungi : Dissertation. / Gasparetti, Chiara.

Espoo : VTT Technical Research Centre of Finland, 2012. 170 p.

Research output: ThesisDissertationCollection of Articles

TY - THES

T1 - Biochemical and structural characterisation of the copper containing oxidoreductases catechol oxidase, tyrosinase, and laccase from ascomycete fungi

T2 - Dissertation

AU - Gasparetti, Chiara

PY - 2012

Y1 - 2012

N2 - Catechol oxidase (EC 1.10.3.1), tyrosinase (EC 1.14.18.1), and laccase (EC 1.10.3.2) are copper-containing metalloenzymes. They oxidise substituted phenols and use molecular oxygen as a terminal electron acceptor. Catechol oxidases and tyrosinases catalyse the oxidation of p-substituted o-diphenols to the corresponding o-quinones. Tyrosinases also catalyse the introduction of a hydroxyl group in the ortho position of p-substituted monophenols and the subsequent oxidation to the corresponding o-quinones. Laccases can oxidise a wide range of compounds by removing single electrons from the reducing group of the substrate and generate free radicals. The reaction products of these oxidases can react further non-enzymatically and lead to formation of polymers and cross-linking of proteins and carbohydrates, in certain conditions. The work focused on examination of the properties of catechol oxidases, tyrosinases and laccases. A novel catechol oxidase from the ascomycete fungus Aspergillus oryzae was characterised from biochemical and structural point of view. Tyrosinases from Trichoderma reesei and Agaricus bisporus were examined in terms of substrate specificity and inhibition. The oxidation capacity of laccases was elucidated by using a set of laccases with different redox potential and a set of substituted phenolic substrates with different redox potential. Finally, an evaluation of the protein cross-linking ability of catechol oxidase from A. oryzae, tyrosinases from T. reesei and A. bisporus and laccases from Trametes hirsuta, Thielavia arenaria, and Melanocarpus albomyces was performed. A novel extracellular catechol oxidase from A. oryzae (AoCO4; UniProtKB: Q2UNF9; Entrez gene ID: 5990879) was chosen for cloning and expression as representative of the newly discovered family of short tyrosinases sequences. AoCO4 gene was heterologously expressed in T. reesei. The protein produced did not show activity on L-tyrosine and 3,4-dihydroxy-L-phenylalanine (L-DOPA), which are typical substrates for tyrosinases. Consequently, the protein was classified as a catechol oxidase. AoCO4 was produced in a bioreactor and the expression resulted in high yields. The purified AoCO4 was partially processed at a Kex2/furin-type protease site and showed a molecular weight of 39.3 kDa. AoCO4 was able to oxidise a limited range of diphenolic compounds, e.g., catechol, caffeic acid, hydrocaffeic acid, and 4-tert-buthylcatechol. AoCO4 oxidised also the monophenolic compounds aminophenol and guaiacol. AoCO4 showed a pH optimum in the acidic range and was observed to be a relatively thermostable enzyme. A crystal structure of AoCO4 was solved at 2.5 Å resolution. AoCO4 was a monomer and the overall structure of AoCO4 was found to be similar to the known structures of catechol oxidases and tyrosinases. A detailed characterisation of the substrate-specificity of the extracellular tyrosinase from T. reesei (TrT) was accomplished and compared to the commercial tyrosinase from A. bisporus (AbT). TrT generally showed lower affinity than AbT on substrates that had a free amino group, such as L-tyrosine, L-DOPA, and YGG tripeptide. The reaction end product of TrT and AbT was studied via mass spectrometry and dopachrome was found to be the only reaction end product of L-DOPA oxidation catalysed by both tyrosinases. Dopachrome produced by AbT-catalysed oxidation of L-DOPA was also shown to inhibit the TrT tyrosinase by an end product inhibition mechanism. Further, when the type of inhibition of potential inhibitors for TrT was analysed with p-coumaric acid and caffeic acid as substrates, potassium cyanide and kojic acid were the strongest inhibitors of TrT. The kinetics of three laccases, with different redox potential (E°), for three p-substituted dimethoxy phenolic substrates (2,6-dimethoxyphenol, syringic acid, and methyl syringate) with different E° were determined at two pHs. The laccases studied were from M. albomyces, T. arenaria, and T. hirsuta. The enzyme from the ascomycete fungus T. arenaria was a novel laccase produced in T. reesei. T. arenaria laccase was purified, and biochemically and structurally characterised. By comparison of the three laccases it could be shown that both the difference in redox potential (?E°) and the pH had an effect on the kinetics. However, the effect of ?E° was found to prevail over that of the pH for substrates with a high E°, such as methyl syringate. All oxidative enzymes studied in this work were also tested for their cross-linking ability, with ?-caseins utilised as a model substrate with and without a cross-linking agent. TrT was found to be the best enzyme for cross-linking of ?-caseins, whereas AbT was found to be the best enzyme for cross-linking ?-caseins in the presence of catechol as a cross-linking agent. Interestingly, AoCO4 was also found to cross-link ?-caseins in the presence of catechol as a cross-linking agent.

AB - Catechol oxidase (EC 1.10.3.1), tyrosinase (EC 1.14.18.1), and laccase (EC 1.10.3.2) are copper-containing metalloenzymes. They oxidise substituted phenols and use molecular oxygen as a terminal electron acceptor. Catechol oxidases and tyrosinases catalyse the oxidation of p-substituted o-diphenols to the corresponding o-quinones. Tyrosinases also catalyse the introduction of a hydroxyl group in the ortho position of p-substituted monophenols and the subsequent oxidation to the corresponding o-quinones. Laccases can oxidise a wide range of compounds by removing single electrons from the reducing group of the substrate and generate free radicals. The reaction products of these oxidases can react further non-enzymatically and lead to formation of polymers and cross-linking of proteins and carbohydrates, in certain conditions. The work focused on examination of the properties of catechol oxidases, tyrosinases and laccases. A novel catechol oxidase from the ascomycete fungus Aspergillus oryzae was characterised from biochemical and structural point of view. Tyrosinases from Trichoderma reesei and Agaricus bisporus were examined in terms of substrate specificity and inhibition. The oxidation capacity of laccases was elucidated by using a set of laccases with different redox potential and a set of substituted phenolic substrates with different redox potential. Finally, an evaluation of the protein cross-linking ability of catechol oxidase from A. oryzae, tyrosinases from T. reesei and A. bisporus and laccases from Trametes hirsuta, Thielavia arenaria, and Melanocarpus albomyces was performed. A novel extracellular catechol oxidase from A. oryzae (AoCO4; UniProtKB: Q2UNF9; Entrez gene ID: 5990879) was chosen for cloning and expression as representative of the newly discovered family of short tyrosinases sequences. AoCO4 gene was heterologously expressed in T. reesei. The protein produced did not show activity on L-tyrosine and 3,4-dihydroxy-L-phenylalanine (L-DOPA), which are typical substrates for tyrosinases. Consequently, the protein was classified as a catechol oxidase. AoCO4 was produced in a bioreactor and the expression resulted in high yields. The purified AoCO4 was partially processed at a Kex2/furin-type protease site and showed a molecular weight of 39.3 kDa. AoCO4 was able to oxidise a limited range of diphenolic compounds, e.g., catechol, caffeic acid, hydrocaffeic acid, and 4-tert-buthylcatechol. AoCO4 oxidised also the monophenolic compounds aminophenol and guaiacol. AoCO4 showed a pH optimum in the acidic range and was observed to be a relatively thermostable enzyme. A crystal structure of AoCO4 was solved at 2.5 Å resolution. AoCO4 was a monomer and the overall structure of AoCO4 was found to be similar to the known structures of catechol oxidases and tyrosinases. A detailed characterisation of the substrate-specificity of the extracellular tyrosinase from T. reesei (TrT) was accomplished and compared to the commercial tyrosinase from A. bisporus (AbT). TrT generally showed lower affinity than AbT on substrates that had a free amino group, such as L-tyrosine, L-DOPA, and YGG tripeptide. The reaction end product of TrT and AbT was studied via mass spectrometry and dopachrome was found to be the only reaction end product of L-DOPA oxidation catalysed by both tyrosinases. Dopachrome produced by AbT-catalysed oxidation of L-DOPA was also shown to inhibit the TrT tyrosinase by an end product inhibition mechanism. Further, when the type of inhibition of potential inhibitors for TrT was analysed with p-coumaric acid and caffeic acid as substrates, potassium cyanide and kojic acid were the strongest inhibitors of TrT. The kinetics of three laccases, with different redox potential (E°), for three p-substituted dimethoxy phenolic substrates (2,6-dimethoxyphenol, syringic acid, and methyl syringate) with different E° were determined at two pHs. The laccases studied were from M. albomyces, T. arenaria, and T. hirsuta. The enzyme from the ascomycete fungus T. arenaria was a novel laccase produced in T. reesei. T. arenaria laccase was purified, and biochemically and structurally characterised. By comparison of the three laccases it could be shown that both the difference in redox potential (?E°) and the pH had an effect on the kinetics. However, the effect of ?E° was found to prevail over that of the pH for substrates with a high E°, such as methyl syringate. All oxidative enzymes studied in this work were also tested for their cross-linking ability, with ?-caseins utilised as a model substrate with and without a cross-linking agent. TrT was found to be the best enzyme for cross-linking of ?-caseins, whereas AbT was found to be the best enzyme for cross-linking ?-caseins in the presence of catechol as a cross-linking agent. Interestingly, AoCO4 was also found to cross-link ?-caseins in the presence of catechol as a cross-linking agent.

KW - Trichoderma reesei

KW - Agaricus bisporus

KW - Aspergillus oryzae

KW - Thielavia arenaria

KW - tyrosinase

KW - catechol oxidase

KW - laccase

KW - purification

KW - characterisation

KW - oxidation capacity

KW - inhibition

KW - three-dimensional structure

KW - cross-linking

M3 - Dissertation

SN - 978-951-38-7934-1

T3 - VTT Science

PB - VTT Technical Research Centre of Finland

CY - Espoo

ER -