A robust metallo-oxidase from the hyperthermophilic bacterium Aquifex aeolicus.
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, McoADeltaP321-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 degrees C and presents remarkable heat stability at 80 and 90 degrees C, with activity lasting for up to 9 and 5 h, respectively. McoA probably contributes to copper and iron homeostasis in A. aeolicus.
Fernandes
André T
AT
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
Soares
Cláudio M
CM
Pereira
Manuela M
MM
Huber
Robert
R
Grass
Gregor
G
Martins
Lígia O
LO
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology*; D052160:Benzothiazoles / Q000378:metabolism; D003001:Cloning, Molecular; D003300:Copper / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism; D006835:Hydrazones / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D042845:Laccase / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D013451:Sulfonic Acids / Q000378:metabolism
D052160:Benzothiazoles; D006835:Hydrazones; D013451:Sulfonic Acids; C009370:syringaldazine; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D003300:Copper; D007501:Iron; D010088:Oxidoreductases; D042845:Laccase