Escherichia coli expression and in vitro activation of a unique ligninolytic peroxidase that has a catalytic tyrosine residue

Yuta Miki, María Morales, Francisco J. Ruiz-Dueñas, María Jesús Martínez, Hiroyuki Wariishi, Angel T. Martínez

Research output: Contribution to journalArticle

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Abstract

Heterologous expression of Trametes cervina lignin peroxidase (LiP), the only basidiomycete peroxidase that has a catalytic tyrosine, was investigated. The mature LiP cDNA was cloned into the pET vector and used to transform Escherichia coli. Recombinant LiP protein accumulated in inclusion bodies as an inactive form. Refolding conditions for its in vitro activation-including incorporation of heme and structural Ca2+ ions, and formation of disulfide bridges-were optimized taking as a starting point those reported for other plant and fungal peroxidases. The absorption spectrum of the refolded enzyme was identical to that of wild LiP from T. cervina suggesting that it was properly folded. The enzyme was able to oxidize 1,4-dimethoxybenzene and ferrocytochrome c confirming its high redox potential and ability to oxidize large substrates. However, during oxidation of veratryl alcohol (VA), the physiological LiP substrate, an unexpected initial lag period was observed. Possible modification of the enzyme was investigated by incubating it with H2O2 and VA (for 30 min before dialysis). The pretreated enzyme showed normal kinetics traces for VA oxidation, without the initial lag previously observed. Steady-state kinetics of the pretreated LiP were almost the same as the recombinant enzyme before the pretreatment. Moreover, the catalytic constant (kcat) for VA oxidation was comparable to that of wild LiP from T. cervina, although the Michaelis-Menten constant (Km) was 8-fold higher. The present heterologous expression system provides a valuable tool to investigate structure-function relationships, and autocatalytic activation of the unique T. cervina LiP.

Original languageEnglish
Pages (from-to)208-214
Number of pages7
JournalProtein Expression and Purification
Volume68
Issue number2
DOIs
Publication statusPublished - Dec 1 2009

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Peroxidase
Tyrosine
Escherichia coli
Enzymes
Trametes
Peroxidases
Basidiomycota
In Vitro Techniques
lignin peroxidase
Inclusion Bodies
Cytochromes c
Heme
Disulfides
Oxidation-Reduction
Dialysis
Complementary DNA
Ions
veratryl alcohol
Proteins

All Science Journal Classification (ASJC) codes

  • Biotechnology

Cite this

Escherichia coli expression and in vitro activation of a unique ligninolytic peroxidase that has a catalytic tyrosine residue. / Miki, Yuta; Morales, María; Ruiz-Dueñas, Francisco J.; Martínez, María Jesús; Wariishi, Hiroyuki; Martínez, Angel T.

In: Protein Expression and Purification, Vol. 68, No. 2, 01.12.2009, p. 208-214.

Research output: Contribution to journalArticle

Miki, Yuta ; Morales, María ; Ruiz-Dueñas, Francisco J. ; Martínez, María Jesús ; Wariishi, Hiroyuki ; Martínez, Angel T. / Escherichia coli expression and in vitro activation of a unique ligninolytic peroxidase that has a catalytic tyrosine residue. In: Protein Expression and Purification. 2009 ; Vol. 68, No. 2. pp. 208-214.
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