Metalloenzymes

Blood is red in vertebrates and blue in molluscs thanks to protein–metal complexes. Metalloproteins are involved in all fundamental biochemical processes including respiration, oxygenic photosynthesis, regulation of transcription and translation, nitrogen fixation, and metabolism of xenobiotics. Metalloenzymes were the first biological catalysts on Earth. Later, when the composition of the Earth's atmosphere changed dramatically due to photosynthesis, the metalloproteins evolved to counter the toxic effects of oxygen and harness it for new physiological functions. Currently, over one-half of all proteins are estimated to bind metals. This article introduces but a small number of metalloprotein families, with emphasis on oxidoreductases, electron-transfer proteins, and photosynthetic complexes.

The PROMISE (Prosthetic centres and metal ions in protein active sites) database aims to gather together comprehensive sequence, structural, functional and bibliographic information on proteins which possess prosthetic centres, with an emphasis on active site structure and function. The database is available on the World Wide Web at http://www.bioinf.leeds.ac.uk/promise/

The PROMISE (Prosthetic centres and metal ions in protein active sites) database project has been launched to gather together comprehensive sequence, structural, functional and bibliographic information on proteins which possess prosthetic centres, with an emphasis on active site structure and function. PROMISE version 1.0 comprises data on iron-containing proteins.

The PROMISE (prosthetic centres and metal ions in protein active sites) database aims to present comprehensive sequence, structural, functional and bibliographic information on metalloproteins and other complex proteins, with an emphasis on active site structure and function. The database is available on the WorldWide Web at http://bioinf.leeds.ac.uk/promise/

We have studied the accessibility of the structural calcium ion in the Burkholderia glumae lipase and the consequences of its removal on the protein conformation by different biophysical techniques (circular dichroism, fluorimetry, and mass spectrometry) and by molecular-dynamics simulations. We show that, in the native protein, calcium is not accessible unless specific flexible loops are displaced, for example, by a temperature increase. Such movements concern the whole calcium-binding pocket and particularly the environment of the coordinating aspartate residue 241. As a consequence of metal depletion the protein unfolds irreversibly and undergoes aggregation. The removal of the metal ion causes major structural transitions and leads to an increase in β-structure, in particular in protein regions that are largely unstructured in the native protein and encompass the calcium coordination residues.

In the present study we show that recombinant bacterial CotA-laccase from Bacillus subtilis is able to decolourise, at alkaline pH and in the absence of redox mediators, a variety of structurally different synthetic dyes. The enzymatic biotransformation of the azo dye Sudan Orange G (SOG) was addressed in more detail following a multidisciplinary approach. Biotransformation proceeds in a broad span of temperatures (30–80 °C) and more than 98% of Sudan Orange G is decolourised within 7 h by using 1 U mL−1 of CotA-laccase at 37 °C. The bell-shape pH profile of the enzyme with an optimum at 8, is in agreement with the pH dependence of the dye oxidation imposed by its acid-basic behavior as measured by potentiometric and electrochemical experiments. Seven biotransformation products were identified using high-performance liquid chromatography and mass spectrometry and a mechanistic pathway for the azo dye conversion by CotA-laccase is proposed. The enzymatic oxidation of the Sudan Orange G results in the production of oligomers and, possibly polymers, through radical coupling reactions. A bioassay based on inhibitory effects over the growth of Saccharomyces cerevisiae shows that the enzymatic bioremediation process reduces 3-fold the toxicity of Sudan Orange G.

In the present study we show that recombinant bacterial CotA-laccase from Bacillus subtilis is able to decolourise, at alkaline pH and in the absence of redox mediators, a variety of structurally different synthetic dyes. The enzymatic biotransformation of the azo dye Sudan Orange G (SOG) was addressed in more detail following a multidisciplinary approach. Biotransformation proceeds in a broad span of temperatures (30–80 °C) and more than 98% of Sudan Orange G is decolourised within 7 h by using 1 U mL−1 of CotA-laccase at 37 °C. The bell-shape pH profile of the enzyme with an optimum at 8, is in agreement with the pH dependence of the dye oxidation imposed by its acid-basic behavior as measured by potentiometric and electrochemical experiments. Seven biotransformation products were identified using high-performance liquid chromatography and mass spectrometry and a mechanistic pathway for the azo dye conversion by CotA-laccase is proposed. The enzymatic oxidation of the Sudan Orange G results in the production of oligomers and, possibly polymers, through radical coupling reactions. A bioassay based on inhibitory effects over the growth of Saccharomyces cerevisiae shows that the enzymatic bioremediation process reduces 3-fold the toxicity of Sudan Orange G.

Prion proteins are infectious agents causing transmissible spongiform encephalopathies in a misfolded protease-resistant form of protein. Human PrP possesses 7 potential copper-binding sites. Notably, four of putative copper-binding sites are located in the octarepeat region (PrP 60-91). Recent studies have shown that peptides derived from human PrP effectively bind Cu2+ to form the Cu-centered catalytic complex required for generation of superoxide by coupling the oxidation of neurotransmitters and their analogues. In this study, we have studied the minimal motifs required for binding of metals within human PrP, by assessing (1) the peptide-dependent quenching of Tb3+ fluorescence and (2) the Cu2+-dependent quenching of intrinsic fluorescence in human PrP octarepeat-derived peptides. Our fluorescent assay with Tb-fluorescence quenching supported the positive role of the His-ended X-X-H motif (in this case P-Q-H tripeptide sequence) being favorably located at C-termini of small peptides rather than His-started H-G-G-G-W motif, is desirable as metal chelating motifs in short peptides. Controversially, the role of His-started motif was supported by the Cu-dependent peptide fluorescence quenching assay. Above data suggested that there are two distinct modes of metal binding to His residues in the octarepeat regions in PrP, possibly by co-ordinations of His-started and His-ended motifs around the target metals depending on the conditions given.