Characterization of sulfhydryl oxidase from Aspergillus tubingensis

Outi Nivala, Greta Faccio, Mikko Arvas, Perttu Permi, Johanna Buchert, Kristiina Kruus, Maija Liisa Mattinen

Research output: Contribution to journalArticleScientificpeer-review

Abstract

CONCLUSIONS: AtSOX (55 kDa) is a fungal secreted flavin-dependent enzyme with good stability to both pH and temperature. A Michaelis-Menten behaviour was observed with reduced glutathione as a substrate. Based on the location of SOX enzyme encoding genes close to NRPSs, SOXs could be involved in the secondary metabolism and act as an accessory enzyme in the production of nonribosomal peptides.

BACKGROUND: Despite of the presence of sulfhydryl oxidases (SOXs) in the secretomes of industrially relevant organisms and their many potential applications, only few of these enzymes have been biochemically characterized. In addition, basic functions of most of the SOX enzymes reported so far are not fully understood. In particular, the physiological role of secreted fungal SOXs is unclear.

RESULTS: The recently identified SOX from Aspergillus tubingensis (AtSOX) was produced, purified and characterized in the present work. AtSOX had a pH optimum of 6.5, and showed a good pH stability retaining more than 80% of the initial activity in a pH range 4-8.5 within 20 h. More than 70% of the initial activity was retained after incubation at 50 °C for 20 h. AtSOX contains a non-covalently bound flavin cofactor. The enzyme oxidised a sulfhydryl group of glutathione to form a disulfide bond, as verified by nuclear magnetic resonance spectroscopy. AtSOX preferred glutathione as a substrate over cysteine and dithiothreitol. The activity of the enzyme was totally inhibited by 10 mM zinc sulphate. Peptide- and protein-bound sulfhydryl groups in bikunin, gliotoxin, holomycin, insulin B chain, and ribonuclease A, were not oxidised by the enzyme. Based on the analysis of 33 fungal genomes, SOX enzyme encoding genes were found close to nonribosomal peptide synthetases (NRPS) but not with polyketide synthases (PKS). In the phylogenetic tree, constructed from 25 SOX and thioredoxin reductase sequences from IPR000103 InterPro family, AtSOX was evolutionary closely related to other Aspergillus SOXs. Oxidoreductases involved in the maturation of nonribosomal peptides of fungal and bacterial origin, namely GliT, HlmI and DepH, were also evolutionary closely related to AtSOX whereas fungal thioreductases were more distant.

Original languageEnglish
Article number15
Number of pages1
JournalBMC biochemistry
Volume18
Issue number1
DOIs
Publication statusPublished - 8 Dec 2017
MoE publication typeA1 Journal article-refereed

Fingerprint

Aspergillus
Enzymes
Glutathione
Gene encoding
Gliotoxin
Fungal Genome
Peptide Synthases
Polyketide Synthases
Thioredoxin-Disulfide Reductase
Secondary Metabolism
Zinc Sulfate
sulfhydryl oxidase
Pancreatic Ribonuclease
Peptides
Fungal Proteins
Dithiothreitol
Accessories
Substrates
Disulfides
Nuclear magnetic resonance spectroscopy

Keywords

  • Aspergillus tubingensis
  • Dithiol oxidase
  • Glutathione oxidation
  • Nonribosomal peptide synthesis
  • Secondary metabolism
  • Secreted sulfhydryl oxidase

Cite this

Nivala, O., Faccio, G., Arvas, M., Permi, P., Buchert, J., Kruus, K., & Mattinen, M. L. (2017). Characterization of sulfhydryl oxidase from Aspergillus tubingensis. BMC biochemistry, 18(1), [15]. https://doi.org/10.1186/s12858-017-0090-4
Nivala, Outi ; Faccio, Greta ; Arvas, Mikko ; Permi, Perttu ; Buchert, Johanna ; Kruus, Kristiina ; Mattinen, Maija Liisa. / Characterization of sulfhydryl oxidase from Aspergillus tubingensis. In: BMC biochemistry. 2017 ; Vol. 18, No. 1.
@article{bfeafa6e5c88435f8cf9c2b4a302d132,
title = "Characterization of sulfhydryl oxidase from Aspergillus tubingensis",
abstract = "CONCLUSIONS: AtSOX (55 kDa) is a fungal secreted flavin-dependent enzyme with good stability to both pH and temperature. A Michaelis-Menten behaviour was observed with reduced glutathione as a substrate. Based on the location of SOX enzyme encoding genes close to NRPSs, SOXs could be involved in the secondary metabolism and act as an accessory enzyme in the production of nonribosomal peptides.BACKGROUND: Despite of the presence of sulfhydryl oxidases (SOXs) in the secretomes of industrially relevant organisms and their many potential applications, only few of these enzymes have been biochemically characterized. In addition, basic functions of most of the SOX enzymes reported so far are not fully understood. In particular, the physiological role of secreted fungal SOXs is unclear.RESULTS: The recently identified SOX from Aspergillus tubingensis (AtSOX) was produced, purified and characterized in the present work. AtSOX had a pH optimum of 6.5, and showed a good pH stability retaining more than 80{\%} of the initial activity in a pH range 4-8.5 within 20 h. More than 70{\%} of the initial activity was retained after incubation at 50 °C for 20 h. AtSOX contains a non-covalently bound flavin cofactor. The enzyme oxidised a sulfhydryl group of glutathione to form a disulfide bond, as verified by nuclear magnetic resonance spectroscopy. AtSOX preferred glutathione as a substrate over cysteine and dithiothreitol. The activity of the enzyme was totally inhibited by 10 mM zinc sulphate. Peptide- and protein-bound sulfhydryl groups in bikunin, gliotoxin, holomycin, insulin B chain, and ribonuclease A, were not oxidised by the enzyme. Based on the analysis of 33 fungal genomes, SOX enzyme encoding genes were found close to nonribosomal peptide synthetases (NRPS) but not with polyketide synthases (PKS). In the phylogenetic tree, constructed from 25 SOX and thioredoxin reductase sequences from IPR000103 InterPro family, AtSOX was evolutionary closely related to other Aspergillus SOXs. Oxidoreductases involved in the maturation of nonribosomal peptides of fungal and bacterial origin, namely GliT, HlmI and DepH, were also evolutionary closely related to AtSOX whereas fungal thioreductases were more distant.",
keywords = "Aspergillus tubingensis, Dithiol oxidase, Glutathione oxidation, Nonribosomal peptide synthesis, Secondary metabolism, Secreted sulfhydryl oxidase",
author = "Outi Nivala and Greta Faccio and Mikko Arvas and Perttu Permi and Johanna Buchert and Kristiina Kruus and Mattinen, {Maija Liisa}",
year = "2017",
month = "12",
day = "8",
doi = "10.1186/s12858-017-0090-4",
language = "English",
volume = "18",
journal = "BMC biochemistry",
issn = "1471-2091",
number = "1",

}

Nivala, O, Faccio, G, Arvas, M, Permi, P, Buchert, J, Kruus, K & Mattinen, ML 2017, 'Characterization of sulfhydryl oxidase from Aspergillus tubingensis', BMC biochemistry, vol. 18, no. 1, 15. https://doi.org/10.1186/s12858-017-0090-4

Characterization of sulfhydryl oxidase from Aspergillus tubingensis. / Nivala, Outi; Faccio, Greta; Arvas, Mikko; Permi, Perttu; Buchert, Johanna; Kruus, Kristiina; Mattinen, Maija Liisa.

In: BMC biochemistry, Vol. 18, No. 1, 15, 08.12.2017.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Characterization of sulfhydryl oxidase from Aspergillus tubingensis

AU - Nivala, Outi

AU - Faccio, Greta

AU - Arvas, Mikko

AU - Permi, Perttu

AU - Buchert, Johanna

AU - Kruus, Kristiina

AU - Mattinen, Maija Liisa

PY - 2017/12/8

Y1 - 2017/12/8

N2 - CONCLUSIONS: AtSOX (55 kDa) is a fungal secreted flavin-dependent enzyme with good stability to both pH and temperature. A Michaelis-Menten behaviour was observed with reduced glutathione as a substrate. Based on the location of SOX enzyme encoding genes close to NRPSs, SOXs could be involved in the secondary metabolism and act as an accessory enzyme in the production of nonribosomal peptides.BACKGROUND: Despite of the presence of sulfhydryl oxidases (SOXs) in the secretomes of industrially relevant organisms and their many potential applications, only few of these enzymes have been biochemically characterized. In addition, basic functions of most of the SOX enzymes reported so far are not fully understood. In particular, the physiological role of secreted fungal SOXs is unclear.RESULTS: The recently identified SOX from Aspergillus tubingensis (AtSOX) was produced, purified and characterized in the present work. AtSOX had a pH optimum of 6.5, and showed a good pH stability retaining more than 80% of the initial activity in a pH range 4-8.5 within 20 h. More than 70% of the initial activity was retained after incubation at 50 °C for 20 h. AtSOX contains a non-covalently bound flavin cofactor. The enzyme oxidised a sulfhydryl group of glutathione to form a disulfide bond, as verified by nuclear magnetic resonance spectroscopy. AtSOX preferred glutathione as a substrate over cysteine and dithiothreitol. The activity of the enzyme was totally inhibited by 10 mM zinc sulphate. Peptide- and protein-bound sulfhydryl groups in bikunin, gliotoxin, holomycin, insulin B chain, and ribonuclease A, were not oxidised by the enzyme. Based on the analysis of 33 fungal genomes, SOX enzyme encoding genes were found close to nonribosomal peptide synthetases (NRPS) but not with polyketide synthases (PKS). In the phylogenetic tree, constructed from 25 SOX and thioredoxin reductase sequences from IPR000103 InterPro family, AtSOX was evolutionary closely related to other Aspergillus SOXs. Oxidoreductases involved in the maturation of nonribosomal peptides of fungal and bacterial origin, namely GliT, HlmI and DepH, were also evolutionary closely related to AtSOX whereas fungal thioreductases were more distant.

AB - CONCLUSIONS: AtSOX (55 kDa) is a fungal secreted flavin-dependent enzyme with good stability to both pH and temperature. A Michaelis-Menten behaviour was observed with reduced glutathione as a substrate. Based on the location of SOX enzyme encoding genes close to NRPSs, SOXs could be involved in the secondary metabolism and act as an accessory enzyme in the production of nonribosomal peptides.BACKGROUND: Despite of the presence of sulfhydryl oxidases (SOXs) in the secretomes of industrially relevant organisms and their many potential applications, only few of these enzymes have been biochemically characterized. In addition, basic functions of most of the SOX enzymes reported so far are not fully understood. In particular, the physiological role of secreted fungal SOXs is unclear.RESULTS: The recently identified SOX from Aspergillus tubingensis (AtSOX) was produced, purified and characterized in the present work. AtSOX had a pH optimum of 6.5, and showed a good pH stability retaining more than 80% of the initial activity in a pH range 4-8.5 within 20 h. More than 70% of the initial activity was retained after incubation at 50 °C for 20 h. AtSOX contains a non-covalently bound flavin cofactor. The enzyme oxidised a sulfhydryl group of glutathione to form a disulfide bond, as verified by nuclear magnetic resonance spectroscopy. AtSOX preferred glutathione as a substrate over cysteine and dithiothreitol. The activity of the enzyme was totally inhibited by 10 mM zinc sulphate. Peptide- and protein-bound sulfhydryl groups in bikunin, gliotoxin, holomycin, insulin B chain, and ribonuclease A, were not oxidised by the enzyme. Based on the analysis of 33 fungal genomes, SOX enzyme encoding genes were found close to nonribosomal peptide synthetases (NRPS) but not with polyketide synthases (PKS). In the phylogenetic tree, constructed from 25 SOX and thioredoxin reductase sequences from IPR000103 InterPro family, AtSOX was evolutionary closely related to other Aspergillus SOXs. Oxidoreductases involved in the maturation of nonribosomal peptides of fungal and bacterial origin, namely GliT, HlmI and DepH, were also evolutionary closely related to AtSOX whereas fungal thioreductases were more distant.

KW - Aspergillus tubingensis

KW - Dithiol oxidase

KW - Glutathione oxidation

KW - Nonribosomal peptide synthesis

KW - Secondary metabolism

KW - Secreted sulfhydryl oxidase

UR - http://www.scopus.com/inward/record.url?scp=85038400841&partnerID=8YFLogxK

U2 - 10.1186/s12858-017-0090-4

DO - 10.1186/s12858-017-0090-4

M3 - Article

VL - 18

JO - BMC biochemistry

JF - BMC biochemistry

SN - 1471-2091

IS - 1

M1 - 15

ER -