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A robust metallo‐oxidase from the hyperthermophilic bacterium Aquifex aeolicus
Author(s) -
Fernandes André T.,
Soares Cláudio M.,
Pereira Manuela M.,
Huber Robert,
Grass Gregor,
Martins Lígia O.
Publication year - 2007
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2007.05803.x
Subject(s) - aquifex aeolicus , multicopper oxidase , oxidase test , enzyme , thermostability , escherichia coli , chemistry , biochemistry , copper , ferrous , methionine , mutant , biology , gene , amino acid , laccase , organic chemistry
The gene, Aquifex aeolicus AAC07157.1, encoding a multicopper oxidase (McoA) and localized in the genome as part of a putative copper‐resistance determinant, has been cloned, over‐expressed in Escherichia coli and the recombinant enzyme purified to homogeneity. The purified enzyme shows spectroscopic and biochemical characteristics typical of the well‐characterized multicopper oxidase family of enzymes. McoA presents higher specificity ( k cat / K m ) for cuprous and ferrous ions than for aromatic substrates and is therefore designated as a metallo‐oxidase. Addition of copper is required for maximal catalytic efficiency. A comparative model structure of McoA has been constructed and a striking structural feature is the presence of a methionine‐rich region (residues 321–363), reminiscent of those found in copper homeostasis proteins. The kinetic properties of a mutant enzyme, McoAΔP321‐V363, deleted in the methionine‐rich region, provide evidence for the key role of this region in the modulation of the catalytic mechanism. McoA has an optimal temperature of 75 °C and presents remarkable heat stability at 80 and 90 °C, with activity lasting for up to 9 and 5 h, respectively. McoA probably contributes to copper and iron homeostasis in A. aeolicus .