Molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the Bacillus subtilis endospore coat

Lígia O. Martins, Cláudio M. Soares, Manuela M. Pereira, Miguel Teixeira, Teresa Costa, George H. Jones, Adriano O. Henriques

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419 Citations (Scopus)

Abstract

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 (TI) and two buried copper centers (T2 and T3). Single amino acid substitutions in the CotA TI 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 ≤pH 3.0 and at pH 7.0 for ABTS or SGZ oxidation, respectively. The apparent Km values for ABTS and SGZ at 37 °C were of 106 ± 11 and 26 ± 2 μM, respectively, with corresponding kcat values of 16.8 ± 0.8 and 3.7 ± 0.1 s-1. Maximal enzyme activity was observed at 75 °C with ABTS as substrate. Remarkably, the coat-associated or the purified enzyme showed a half-life of inactivation at 80 °C of about 4 and 2 h, respectively, indicating that CotA is intrinsically highly thermostable.

Original languageEnglish
Pages (from-to)18849-18859
Number of pages11
JournalJournal of Biological Chemistry
Volume277
Issue number21
DOIs
Publication statusPublished - 24 May 2002

Keywords

  • Substitution reactions
  • Substrates
  • Amino acids
  • Bacteria
  • Escherichia coli
  • Organic compounds
  • Oxidation
  • pH effects
  • Polypeptides

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