tailieunhanh - Báo cáo khoa học: A robust metallo-oxidase from the hyperthermophilic bacterium Aquifex aeolicus

The gene,Aquifex , encoding a multicopper oxidase (McoA) and localized in the genome as part of a putative copper-resistance determinant, has been cloned, over-expressed inEscherichia coliand the recombinant enzyme purified to homogeneity. | ỊFEBS Journal A robust metallo-oxidase from the hyperthermophilic bacterium Aquifex aeolicus Andre T. Fernandes1 Claudio M. Soares1 Manuela M. Pereira1 Robert Huber2 Gregor Grass3 and Ligia O. Martins1 1 Institute de Tecnologia Quimica e Biologica Universidade Nova de Lisboa Oeiras Portugal 2 Lehrstuhl fur Mikrobiologie Universitat Regensburg Germany 3 Institute fur Biologie Mikrobiologie Marthin Luther Universitat Halle Germany Keywords Aquifex aeolicus copper and iron homeostasis hyperthermophilic bacteria metallo-oxidase multicopper oxidases Correspondence L. O. Martins Instituto deTecnologia Quimica e Biologica Universidade Nova de Lisboa Avenida da Republica 2781-901 Oeiras Portugal Fax 351 214411277 Tel 351 214469534 E-mail lmartins@ Website http martins Received 22 January 2007 revised 13 March 2007 accepted 22 March 2007 doi The gene Aquifex aeolicus 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 kcat Km 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 McoAAP321-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 .