Molecular and Biochemical Characterization of a Highly Stable Bacterial Laccase That Occurs as a Structural Component of theBacillus 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 overproducingEscherichia 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 K m values for ABTS and SGZ at 37 °C were of 106 ± 11 and 26 ± 2 μm, respectively, with correspondingk cat 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.

• Publication year

  2002

• Venue

  Journal of Biological Chemistry

• Publication date

  2002-05-24

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1074/JBC.M200827200PMID 11884407
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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