Escherichia coli expression and in vitro activation of a unique ligninolytic peroxidase that has a catalytic tyrosine residue.
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 (k(cat)) for VA oxidation was comparable to that of wild LiP from T. cervina, although the Michaelis-Menten constant (K(m)) 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.
Miki
Yuta
Y
Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain.
Morales
María
M
Ruiz-Dueñas
Francisco J
FJ
Martínez
María Jesús
MJ
Wariishi
Hiroyuki
H
Martínez
Angel T
AT
D000595:Amino Acid Sequence; D001592:Benzyl Alcohols / Q000378:metabolism; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D055550:Protein Stability; D055454:Trametes / Q000201:enzymology* / Q000235:genetics; D014443:Tyrosine / Q000378:metabolism*
D001592:Benzyl Alcohols; D014443:Tyrosine; D006861:Hydrogen Peroxide; D010544:Peroxidases; C042858:lignin peroxidase; C042197:veratryl alcohol