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CotA, a Multicopper Oxidase from Bacillus pumilus WH4, Exhibits Manganese-Oxidase Activity

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      Abstract

      Multicopper oxidases (MCOs) are a family of enzymes that use copper ions as cofactors to oxidize various substrates. Previous research has demonstrated that several MCOs such as MnxG, MofA and MoxA can act as putative Mn(II) oxidases. Meanwhile, the endospore coat protein CotA from Bacillus species has been confirmed as a typical MCO. To study the relationship between CotA and the Mn(II) oxidation, the cotA gene from a highly active Mn(II)-oxidizing strain Bacillus pumilus WH4 was cloned and overexpressed in Escherichia coli strain M15. The purified CotA contained approximately four copper atoms per molecule and showed spectroscopic properties typical of blue copper oxidases. Importantly, apart from the laccase activities, the CotA also displayed substantial Mn(II)-oxidase activities both in liquid culture system and native polyacrylamide gel electrophoresis. The optimum Mn(II) oxidase activity was obtained at 53°C in HEPES buffer (pH 8.0) supplemented with 0.8 mM CuCl 2. Besides, the addition of o-phenanthroline and EDTA both led to a complete suppression of Mn(II)-oxidizing activity. The specific activity of purified CotA towards Mn(II) was 0.27 U/mg. The K m, V max and k cat values towards Mn(II) were 14.85±1.17 mM, 3.01×10 −6±0.21 M·min −1 and 0.32±0.02 s −1, respectively. Moreover, the Mn(II)-oxidizing activity of the recombinant E. coli strain M15-pQE- cotA was significantly increased when cultured both in Mn-containing K liquid medium and on agar plates. After 7-day liquid cultivation, M15-pQE- cotA resulted in 18.2% removal of Mn(II) from the medium. Furthermore, the biogenic Mn oxides were clearly observed on the cell surfaces of M15-pQE- cotA by scanning electron microscopy. To our knowledge, this is the first report that provides the direct observation of Mn(II) oxidation with the heterologously expressed protein CotA, Therefore, this novel finding not only establishes the foundation for in-depth study of Mn(II) oxidation mechanisms, but also offers a potential biocatalyst for Mn(II) removal.

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      Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.

      A discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system for the separation of proteins in the range from 1 to 100 kDa is described. Tricine, used as the trailing ion, allows a resolution of small proteins at lower acrylamide concentrations than in glycine-SDS-PAGE systems. A superior resolution of proteins, especially in the range between 5 and 20 kDa, is achieved without the necessity to use urea. Proteins above 30 kDa are already destacked within the sample gel. Thus a smooth passage of these proteins from sample to separating gel is warranted and overloading effects are reduced. This is of special importance when large amounts of protein are to be loaded onto preparative gels. The omission of glycine and urea prevents disturbances which might occur in the course of subsequent amino acid sequencing.
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        Geomicrobiology of manganese(II) oxidation.

        Mn(II)-oxidizing microbes have an integral role in the biogeochemical cycling of manganese, iron, nitrogen, carbon, sulfur, and several nutrients and trace metals. There is great interest in mechanistically understanding these cycles and defining the importance of Mn(II)-oxidizing bacteria in modern and ancient geochemical environments. Linking Mn(II) oxidation to cellular function, although still enigmatic, continues to drive efforts to characterize manganese biomineralization. Recently, complexed-Mn(III) has been shown to be a transient intermediate in Mn(II) oxidation to Mn(IV), suggesting that the reaction might involve a unique multicopper oxidase system capable of a two-electron oxidation of the substrate. In biogenic and abiotic synthesis experiments, the application of synchrotron-based X-ray scattering and spectroscopic techniques has significantly increased our understanding of the oxidation state and relatively amorphous structure (i.e. delta-MnO(2)-like) of biogenic oxides, providing a new blueprint for the structural signature of biogenic Mn oxides.
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          Molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the Bacillus subtilis endospore coat.

          The Bacillus subtilis endospore coat protein CotA shows laccase activity. By using comparative modeling techniques, we were able to derive a model for CotA based on the known x-ray structures of zucchini ascorbate oxidase and Cuprinus cereneus laccase. This model of CotA contains all the structural features of a laccase, including the reactive surface-exposed copper center (T1) and two buried copper centers (T2 and T3). Single amino acid substitutions in the CotA T1 copper center (H497A, or M502L) did not prevent assembly of the mutant proteins into the coat and did not alter the pattern of extractable coat polypeptides. However, in contrast to a wild type strain, both mutants produced unpigmented colonies and spores unable to oxidize syringaldazine (SGZ) and 2'2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). The CotA protein was purified to homogeneity from an overproducing Escherichia coli strain. The purified CotA shows an absorbance and a EPR spectra typical of blue multicopper oxidases. Optimal enzymatic activity was found at < or =pH 3.0 and at pH 7.0 for ABTS or SGZ oxidation, respectively. The apparent K(m) values for ABTS and SGZ at 37 degrees C were of 106 +/- 11 and 26 +/- 2 microm, respectively, with corresponding k(cat) values of 16.8 +/- 0.8 and 3.7 +/- 0.1 s(-1). Maximal enzyme activity was observed at 75 degrees C with ABTS as substrate. Remarkably, the coat-associated or the purified enzyme showed a half-life of inactivation at 80 degrees C of about 4 and 2 h, respectively, indicating that CotA is intrinsically highly thermostable.
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            Author and article information

            Affiliations
            [1 ]State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, People’s Republic of China
            [2 ]Key Laboratory of Arable Land Conservation, Ministry of Agriculture, College of Resources and Environment, Huazhong Agricultural University, Wuhan, Hubei, People’s Republic of China
            Instituto de Tecnologia Quimica e Biologica, Portugal
            Author notes

            Competing Interests: The authors have declared that no competing interests exist.

            Conceived and designed the experiments: FL JH. Performed the experiments: JS PB TB LD HW. Analyzed the data: JS PB TB LD. Contributed reagents/materials/analysis tools: JH. Wrote the paper: JS FL JH.

            Contributors
            Role: Editor
            Journal
            PLoS One
            PLoS ONE
            plos
            plosone
            PLoS ONE
            Public Library of Science (San Francisco, USA )
            1932-6203
            2013
            5 April 2013
            : 8
            : 4
            23577125 3618234 PONE-D-12-34818 10.1371/journal.pone.0060573

            This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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            Pages: 13
            Funding
            This work was supported by the Chinese National Natural Science Funds (grant 40830527), the National Basic Research Program of China (973 Program, grant 2010CB126105), the National High Technology Research and Development Program of China (863 project, grant 2011AA10A205), and the Fundamental Research Funds for Central Universities of China (grant 2011PY092). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
            Categories
            Research Article
            Biology
            Biochemistry
            Enzymes
            Enzyme Kinetics
            Bioinorganic Chemistry
            Microbiology
            Bacteriology
            Bacterial Biochemistry
            Applied Microbiology
            Industrial Microbiology
            Chemistry
            Chemical Biology
            Environmental Chemistry
            Pollutants
            Heavy Metals
            Earth Sciences
            Environmental Sciences
            Environmental Engineering
            Geochemistry
            Biogeochemistry

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